Dna and rna conformational switches as sensitive electronic sensors of analytes

ABSTRACT

The electrical conductivity of DNA and other oligonucleotide constructs is dependent on its conformational state. Such a dependence may be harnessed for the electronic sensing of external analytes, for instance, adenosine or thrombin. Such a DNA sensor incorporates an analyte receptor, whose altered conformation in the presence of bound analyte switches the conformation, and hence, the conductive path between two oligonucleotide stems, such as double-helical DNA. Two distinct designs for such sensors are described that permit significant electrical conduction through a first or “detector” double-helical stem only in the presence of the bound analyte. In the first design, current flows through the analyte receptor itself whereas, in the second, current flows in a path adjacent to the receptor. The former design may be especially suitable for certain categories of analytes, including heterocycle-containing compounds such as adenosine, whereas the latter design should be generally applicable to the detection of any molecular analyte, large or small, such as the protein thrombin. Since analyte detection in these DNA sensors is electronic, the sensors may be used in rapid and automated chip-based detection of small molecules as well as of proteins and other macromolecules.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/507,387 filed 11 Mar. 2003, which claims the benefit of thefiling date of U.S. provisional patent application No. 60/362,928 filed11 Mar. 2002.

TECHNICAL FIELD

This application relates to biosensors comprising DNA or otheroligonucleotides for electronically detecting the presence of analytes.The sensors rely on changes in DNA conformation induced by binding of atarget analyte to a receptor site of the sensor. The conformationalchanges modulate changes in charge transfer through the DNA, which isdetectable either directly or indirectly.

BACKGROUND

Contemporary research in the life sciences, including recent advances ingenomics and proteomics, has led to the discovery of thousands ofproteins with potential diagnostic and/or therapeutic significance. Forexample, some of these proteins are biological markers for organactivity, disease processes, or drug action. The ability to monitorslight differences in the amounts of these proteins as well as otherbiological macromolecules, in the smallest possible detection volumes,down to the level of single cells, is of utmost importance not only forproteomics research but also for biomedical diagnostics in general.^(1a)To date, antibody-based immunological assays (e.g., ELISA) are the mostcommonly used diagnostic methods for protein detection; typically, theyare not as sensitive or specific as methods for detection of specificnucleic acid sequences, DNA microarrays for instance.^(1b) Thus, thereis a need for DNA-based technologies capable of detecting proteins invery low quantities.

Despite a lack of complete understanding of the mechanistic details ofelectron transfer through DNA, long-range electron transfer indouble-stranded DNA is generally believed to be the result of amulti-step hopping reaction¹⁻². The consensus view is that a continuousbase-stacking throughout the DNA duplex is essential for efficientcharge transfer. It has been shown that efficiency of charge transfer isreduced in duplexes containing mismatches³⁻⁵ and bulges⁶. Proteins thatbind and disrupt continuous base-stacking in duplex DNA also reduce theefficiency of electron transfer past the site of helix disruption⁷⁻⁸.Despite the importance of a continuous base stack, not all perturbationsto the helix prevent charge transfer, as it has been observed in helicescontaining abasic sites⁹ and through short, single stranded overhangs¹⁰.However, even these latter structures are believed to base-stack to someextent, which permits charge transfer through them.

Detection of charge transfer in DNA has been detected both directly andindirectly. Dehydrated DNA duplexes¹¹ or DNA fibers^(12a,b) positionedbetween metal electrodes have had their conductivity measured directly.Indirect measurement of DNA conductivity has been made in aqueoussolution, after inducement of charge transfer with a photoexcitablemoiety (such as anthraquinone¹³, or rhodium(III) complexes with aromaticligands¹⁴). The photoexcitable moiety is attached to one end of a duplexsuch that it lies in intimate contact with the π-stack of the DNA basepairs. The photo-excited states of anthraquinone and rhodium(III)complexes are powerful oxidising agents, and are able to collectelectrons from guanines (via generation of a mobile radical cation, orelectron hole) within the DNA duplex, from reported distances of upto >200 Å away from the ligand^(15a,b)). According to the putative“multi-step hopping” mechanism referred to above, the radical cationmoves from guanine to guanine (guanine is the base with the lowestionization potential). A guanine upon which the mobile radical cation istransiently localized is somewhat susceptible to reaction with water anddissolved oxygen, leading to the formation of oxidation products such asdiaminooxazalone and 2-aminoimidazalone¹⁶. As described herein, theposition of the latter products along a DNA strand can readily bedetected by sequencing gel-electrophoresis, since these products arebase-labile and cause site-specific strand breakage on being treatedwith hot piperidine.

Despite disagreements on the precise mode of charge transfer within DNAduplexes, investigators are in agreement that the electricalconductivity of DNA is dependent on its conformationalstate—specifically, on the integrity of its π-stacking. While much ofthe research on DNA conduction has focused on “static” or relativelyimmobile DNA structures, the purpose of the present invention is toexploit changes in the conductivity of DNA, dependent on changes in itsconformational state, to provide information about the DNA'senvironment—such as the presence or absence of a specific analyte. Inother words, if conformational change in the DNA results from thebinding of a particular analyte, then this should correlate with achange in the DNA's conductivity, providing the basis for an analytesensing device constructed from DNA or other oligonucleotides.

In nature, DNA is known to bind a variety of small molecule as well asmacromolecular ligands. However, recent innovations in in vitroselection (SELEX) methods have resulted in DNA (as well as RNA)“aptamer” sequences, which are capable of specifically binding a varietyof molecular species, including many that normally do not interact withDNA or RNA¹⁷. Such aptamer oligonucleotides frequently exhibitinduced-fit folding behaviour (reviewed by Hermann & Patel¹⁸), wherebythe aptamer itself, largely unstructured in solution, undergoessignificant compaction and structural stabilization upon binding itscognate ligand. Due to the ease with which novel, made-to-order aptamerscan be selected from large, random sequence DNA and RNA libraries, andtheir generally impressive selectivity and affinity, they are widelyregarded as ideal recognition elements for biosensor applications.^(18a)

Barton and colleagues¹⁹ have reported the electronic detection of aDNA-binding protein, HhaI methyltransferase, by virtue of the protein'sinterference in the charge conduction path of a duplex DNA. HhaImethylase works by binding to a target G*CGC site on a double-helix, andextruding the target cytosine base (marked with an asterisk, above) outof the helix in order to methylate it. This extrusion naturally disruptsthe conduction path through the helix and, thus, the level of conductionthrough the helix. While this approach demonstrates protein-modulationof charge-transfer through DNA, it requires the selection of a proteincapable of extruding a base out from the DNA helix and is therefore notof general application. For example, unlike the present invention, themethod could not be readily extended as a means for the detection of anyprotein, large or small, DNA-binding or not.

It is also known in the prior art to detect conformational change induplex DNA by binding of divalent metal ions. Lee and colleagues²⁰ havereported a methodology for the electronic detection of a DNA-bindingprotein. Following the binding of the protein to its binding site upon aDNA duplex, the DNA is converted to a metal-bound form (“M-DNA”), with asignificantly higher conductivity than that of standard B-DNA. Thepresence of the bound protein, however, interferes with M-DNA formationby its binding site, and therefore affects the overall conductivity ofthe duplex. While this approach is promising, the efficacy of thismethod for use in the detection of proteins that do not naturally bindto DNA, or which bind to non-duplex elements of DNA or RNA, has not yetbeen reported.

The need has therefore arisen for improved biosensors of generalapplication for analyte detection, and in particular, biosensors forrapidly detecting proteins at low concentrations in biological fluids.Since the detection means is electronic, the potential exists for use ofsuch sensors for rapid and automated chip-based detection of smallmolecules as well as of proteins and other macromolecules. The sensorsare also potentially useful as nanoelectronic switches and junctiondevices simulating solid state electronic logic gates.

SUMMARY OF INVENTION

In accordance with the invention, an analyte sensor comprising a firstoligonucleotide stem, a second oligonucleotide stem, and a receptor sitecapable of binding the analyte is provided. The receptor site isoperatively connected to the first and second stems. The sensor isalterable between a first conformational state substantially impedingcharge transfer between the first and second stems and a secondconformational state permitting charge transfer between the first andsecond stems. The sensor switches between the first conformational stateand the second conformational state when the analyte binds to thereceptor site.

The charge may be conducted between the first and second stems throughthe receptor site in the second conformational state. Alternatively, thereceptor site may be removed from the conduction path between the firstand second stems such that the receptor does not function as a conductorin either of the first and second conformational states.

In one embodiment of the invention the sensor switches from the firstconformational state to the second conformational state (i.e. resultingin increased charge transfer) when the analyte binds to the receptorsite. In another embodiment, the sensor switches from the secondconformational state to the first conformational state (i.e. resultingin decreased charge transfer) when the analyte binds to the receptorsite. In either case, the change in charge transfer is measurable todetect the presence of the analyte. The receptor site may be configuredto bind to an analyte which does not ordinarily bind to DNA.

The receptor site may comprise a nucleic acid apatmer selected forbinding affinity to a target analyte. The first and secondoligonucleotide stems may each comprise helical DNA. In the firstconformational state the base pairing of the helical DNA may bedisrupted in a switch domain located at or near the receptor site in thefirst conformational state. When the analyte binds to the receptor, aconformational change to the second conformation state occurs, resultingin removal or lessening of the base pairing disruption. This in turnresults in increased charge transfer between the first and second stemsin this embodiment.

In one embodiment of the invention a detector may be electricallycoupled to the first stem and may directly measure the change in chargetransfer through the sensor resulting from analyte binding. The detectormay, for example, comprise a conductor or semi-conductor chip.

In other embodiments of the invention the change in charge transferresulting from binding of the analyte to the sensor may be detectedindirectly. For example, a charge flow inducer may be coupled to one ofthe first and second stems for triggering charge flow in at least one ofthe first and second stems. The charge flow inducer may comprise, forexample, a photoexcitable moiety, such as antraquinone or rhodium (III),coupled to the second stem. In these examples, the photo-excited statesof such compounds are oxidizing agents which cause a net flow ofelectrons toward the photoexcitable moiety. If the sensor is in thesecond conformational state this process results in the formation ofoxidizing products which may be detected, for example, bygel-electrophoresis. For example, the gel-electrophoresis may identifydamage to specific guanine residues concomitant with electron donation(and hence indicative of charge transfer between the first and secondstems).

In one embodiment of the invention the sensor may comprise a thirdoligonucleotide stem which includes the receptor site. The first, secondand third stems may be connected together at a three-way junction. Thebinding of the analyte to the receptor site on the third stem modulatescharge transfer between the first and second stems. At least one of thestems may include unpaired nucleotides in the first conformational state(e.g. non-Watson-Crick base pairs located near the three-way junction).Further, the sensor could optionally include a fourth oligonucleotidestem connected to the first, second and third stems at a four-wayjunction. The stems could each comprise helical DNA. Sensors constructedfrom other multi-stem nucleotide sensors (e.g. five, six or morenucleotide stems) may be configured in the same manner. In each case aconformational change to at least some of the stems occurs upon bindingof the target analyte, which is detectable by directly or indirectlyidentifying a change in charge conduction.

In a further alternative embodiment of the invention pairs of sensorsmay be configured to simulate digital electronics logic gates. Eachsensor is notionally operable in one of two operating states, namely“conducting” (i.e. “on”) and “nonconducting” (i.e. “off”). For example,the sensor may comprise two separate receptor sites and may only switchbetween the first and second conformational states when both of thereceptors bind to their respective target analytes.

A method for detecting the presence of an analyte is also disclosed. Themethod includes the steps of (a) providing a sensor comprising first andsecond oligonucleotide stems and a receptor site operatively connectedto the first and second stems and capable of binding the analyte asdescribed above; (b) inducing a net charge flow in one of the first andsecond stems of the detector; and (c) detecting any change in chargetransfer between the first and second stems upon binding of the analyteto the receptor.

The step of detecting changes in electrical charge transfer may includeelectrically coupling a conductor or semi-conductor detector to thefirst stem and measuring the change in charge transfer resulting fromanalyte binding. Alternatively, a charge flow inducer coupled to thesensor could be triggered to produce an oxidizing agent. According tothis protocol, the sensor is then tested for oxidation products. Forexample, the sensor could be heated in the presence of piperidine andthe formation of oxidation products, and the specific site of DNAcleavage, may be detected by gel electrophoresis. The direct or indirectdetection of changes in electrical charge transfer may also beaccomplished by other means known in the art, such as fluorescencequenching.

BRIEF DESCRIPTION OF DRAWINGS

In drawings which describe embodiments of the invention, but whichshould not be construed as restricting the spirit or scope of theinvention in any way,

FIG. 1( a) is a schematic view of the Applicant's sensor design in the“coupled receptor” or “coupled ligand” embodiment. The sensor is alteredfrom a first conformational state (left) to a second conformationalstate (right) upon binding of an analyte (e.g. a ligand) to permitcharge transfer in the direction indicated by arrows.

FIG. 1( b) is a schematic view of the Applicant's sensor design in the“integrated receptor” or “integrated ligand” embodiment. As in FIG. 1(a), the sensor is altered from a first conformational state (left) to asecond conformational state (right) upon binding of an analyte (e.g.ligand).

FIG. 2( a) illustrates electrical conduction through a conventional DNAdouble helix having an anthraquinone moiety covalently tethered thereto.

FIG. 2( b) illustrates a sensor incorporating two unpaired nucleotidesat a 3-way junction in accordance with one embodiment of the invention.

FIG. 2( c) is a schematic view of a DNA construct having a standard3-way junction.

FIG. 3( a) is a schematic view of a RNA/DNA heteroduplex.

FIG. 3( b) is a schematic view of a mixed sensor comprising separate RNAand DNA strands.

FIGS. 3( c) and 3(d) are schematic views of sensors having 4-wayjunctions.

FIG. 4( a) is a schematic view of a protein-detecting sensorillustrating detection by physical interference.

FIG. 4( b) is a schematic view of a protein-detecting sensorillustrating detection by adaptive binding.

FIG. 5 is a schematic view of sensor configured to detect nucleic acids.

FIG. 6 is a schematic view of a sensor designed to simulate an “AND”logic gate.

FIG. 7 is a schematic view of a sensor designed to simulate a “NAND”logic gate.

FIG. 8 shows a sensor which may be switched from an “ON” to an “OFF”logic state upon binding of a target analyte.

FIG. 9 shows a sensor which may be switched from an “OFF” to an “ON”logic state upon binding of a target analyte.

FIG. 10 is a schematic view of a process for in vitro selection ofsensors specific for a particular analyte.

FIG. 11( a) is a schematic view of the Applicant's sensor design in the“coupled receptor” or “coupled ligand” embodiment. The sensor is alteredfrom a first conformational state (left) to a second conformational sate(right) upon binding of an analyte to permit charge transfer in thedirection indicated by arrows. [we may omit this figure—it is equivalentto FIG. 1( a)]

FIG. 11( b) is a schematic view of the Applicant's sensor design in anembodiment wherein the coupled ligand sensor is immobilized on a chipfor the direct electronic detection of analyte. Ferrocene (Fc) iscovalently attached to the end of the reporter stem for electrochemicalmonitoring of the binding event, while the end of the detector stem ismodified for immobilization on the electrode.

FIG. 12 is a schematic view showing the design of “integrated-ligand”and “coupled-ligand” sensors for detecting the analyte adenosine. In theabsence of analyte, both sensors adopt open, unstructured conformations,which only allow charge transfer (indicated by arrows) in theoligonucleotide stem conjugated to the anthraquinone (AQ) moiety.Adenosine binding induces the folding and compaction of the adenosineaptamer, facilitating charge transfer from the detector to the AQ stems.

FIG. 13 compares the nucleotide sequence of a control duplex with theaptamer domain of the analyte sensor of FIG. 12 (showing two boundadenosines). The extent of the aptamer domain is indicated (shown asboxed), with the two bound adenosines shown as outlined ‘A’s. “D” and“P” indicate guanine doublets distally and proximally located,respectively, relative to the covalently conjugated anthraquinone (AQ)moiety.

FIG. 14( a) comprise phosphorimager traces of strand-cleavage data fromthe “integrated-ligand” sensor (lanes 8-14), and, from its duplexcontrol (lanes 1-7). “P” and “D” indicate the positions of the proximaland distal guanine doublets shown in FIG. 13. Lanes 4 and 11 show thecontrol duplex and integrated sensor constructs photo-irradiated in the‘Mg—Na’ buffer (50 mM Tris-Cl, pH 7.9, 2.5 mM MgCl₂, 100 mM NaCl and 0.1mM EDTA) with no added adenosine. Lanes 5 and 12 included 2.5 mMadenosine, and lanes 6 and 13 included 2.5 mM uridine. Lanes 3 and 10show background piperidine cleavage (using the same conditions as withthe other samples) for non-photoirradiated (“dark”) controls for theduplex construct and for the AQ-labeled sensor construct, respectively.Lanes 1 and 8 show constructs that were neither irradiated norpiperidine-treated, while lanes 2 and 9 show constructs that werephoto-irradiated but not piperidine treated. Lanes 7 and 14 showcontrols were ³²P-end labeled constructs lacking AQ werephoto-irradiated in the presence of unlabeled constructs possessing theAQ-functionality. Maxam-Gilbert sequencing reactions were used togenerate the “G” and “C+T” ladders. All photo-iradiation was with a 366nm low-pressure lamp for 90 minutes (45 minutes for double-strandedcontrols) at 18° C., from a distance of 4 cm. Samples were thenpiperidine treated and run on a 12% sequencing gel (11% for doublestranded control).

FIG. 14( b) are phosphorimager traces as in FIG. 14A but carried out inMg buffer (50 nM Tris-Cl, pH 7.9, 2.5 mM MgCl₂, and 0.1 mM EDTA) withphoto-irradiation of 120 minutes at 18° C.

FIG. 15 is a graph showing adenosine-dependence of cleavage at thedistal guanines of the “integrated-ligand” sensor construct. Samples ofthe sensor construct (0.5 μM) were photo-irradiated for 90 minutes at18° C. in Mg buffer containing or not containing Na⁺, in the presence ofvarious adenosine concentrations. Following irradiation, samples werepiperidine treated and loaded on sequencing gels. Strand cleavage,quantitated in a phosphorimager, were corrected against ‘dark,’non-irradiated, controls and normalized for the maximal observedcleavage.

FIG. 16( a) shows the structure and sequence of the “coupled-ligand”sensor. The ATP aptamer domain is indicated as boxed, while the twobound adenosines are indicated by outlined ‘A’s. Guanine doublets in the5′-32P-end labeled strand used to monitor charge transfer to the Sensorand Detector stems are indicated as “x”, “y”, and “z”. The AoG mismatchat the junction was used since it gave superior results relative toWatson-Crick base pairs at that position. The arrow, on an adenine atthe junction, indicates an adenine that showed an unusually highcleavage (see FIG. 16( b), lane 4, below).

FIG. 16( b) are Phosphorimager traces of strand-cleavage data from the“coupled-ligand” sensor construct, irradiated at 18° C. for 180 minutesin the ‘Mg—Na’ buffer. Lanes 3-5 show cleavage results in the presenceof 2.5 mM uridine (lane 3); 2.5 mM adenosine (lane 4); and, buffer alone(lane 5). Lanes 1 and 2 show the Maxam-Gilbert “G” and “C+T” ladders,respectively. Lane 6 shows the background piperidine cleavage of thenon-irradiated construct.

FIG. 17 is a schematic view of the sequences and secondary structures ofembodiments of the Applicant's coupled ligand sensor design tested forthrombin detection. Guanine triplets (“D”) in the 5′-32P-end labeledstrand were used to biochemically monitor charge transfer to the aptamerand/or detector stems from the reporter stem upon photo-excitation ofthe AQ.

FIG. 18 shows phosphorimager traces of strand-cleavage data from theembodiments of the Applicant's coupled ligand sensor design that werecandidates for detecting thrombin. “A” and “D” indicate the positions ofthe guanine doublets in the aptamer and the triple in the detector stemshown in FIG. 17.

FIG. 19 shows the relative biochemical signal (guanine damage at thedetector stem G triplet) (ΔI/I) of the exemplary 3WJ-3 sensor as afunction of protein concentration. The dotted line is to guide the eyesonly.

FIG. 20 shows the design of an embodiment in which “coupled-ligand”sensors are surface-bound to a chip for the electrochemical detection ofthrombin. (a) The secondary structure of the sensor; (b) a purelyWatson-Crick base-paired double helix used as a control.

FIG. 21 shows the electrochemical response of the surface-bound sensorsin the presence of different concentrations of thrombin.

FIG. 22 shows the performance of the immobilized “coupled-ligand” sensorfor the electrochemical detection of thrombin. (a) The sensor response,i.e., the increase in the reduction current upon incubation withthrombin at various concentrations. (b) The linear dependence of sensorsignal on thrombin concentrations (˜) in the range of 0-100 pM. Thesensor response in interferences of 80 pM BSA (

), Avidin (″), IgA (−) or IgG (q) was also shown in this plot.

FIG. 23 shows the interference of serum on the immobilized “coupledligand” sensor for the electrochemical detection of thrombin.

DESCRIPTION 1.0 Description of Alternative Embodiments

Throughout the following description specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practiced without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the present invention.Accordingly, the specification and drawings are to be regarded in anillustrative, rather than a restrictive, sense.

As used in this patent specification, the following terms shall have thefollowing respective meanings:

“analyte” means a molecular entity capable of binding to a receptor.Analyte may include chemical compounds, such as hormones or drugs,antigens, metabolic cofactors, nucleotides, nucleic acid segments,ligands, peptides, proteins, carbohydrates, fats or any other organic orinorganic materials capable of binding to a receptor.

“aptamer” means a single or multi-stranded non-naturally occurringnucleotide sequence that functions as a receptor. Aptamers may beidentified by in vitro selection methods such as SELEX.

“oligonucleotide stem” means singly or multi-stranded RNA, DNA ornucleic-acid like molecules capable of base pairing and permittingcharge conduction under suitable conditions. The term includes stemscomposed of non-naturally occurring nucleic acid analogues, modifiednucleic acids, non-standard nucleic acids and composite DNA/RNAconstructs.

“receptor” means a site on a sensor capable of binding an analyte. Areceptor may comprise nucleic acids, proteins, other organic orinorganic materials, or combinations thereof. The term receptor includesnaturally occurring receptor sites, aptamers and rationally designedartificial receptor sites created de novo other than by SELEX selection.

This application relates to sensors 10 for electrically sensing thepresence and concentration of analytes 12. As described below, sensors10 may be configured to bind to a wide range of target analytes 12, suchas antigens associated with particular disease states.

As shown generally in FIG. 1( a), sensor 10 includes a firstoligonucleotide stem 14 and a second oligonucleotide stem 16 which areconnected together at a junction 18. Stems 14, 16 may consist of doublehelical DNA, for example. In other embodiments oligonucleotide stems 14,16 may comprise other nucleic acid constructs as defined above,including constructs containing synthetic or modified nucleic acidresidues capable of base pairing.

Sensor 10 also includes a receptor 20 which forms part of junction 18 oris located proximate thereto. Receptor 20 may consist of any organic orinorganic site capable of binding to an analyte. By way of example,receptor 20 may consist of a nucleic acid aptamer sequence selected tobind to a target analyte.

In the illustrated embodiment first stem 14 functions as an electrondonor and second stem 16 functions as an electron sink (although thereverse configuration would function similarly so long as a net chargetransfer in either direction is established). As described in detailbelow, sensor 10 is alterable between a first conformational state shownon the left side in FIG. 1( a) where the structure of junction 18substantially impedes charge transfer between the first and second stems14, 16, and a second conformational state shown on the right side inFIG. 1( b) permitting charge transfer through junction 18 between firstand second stems 14, 16. Sensor 10 switches between the first and secondconformational states when analyte 12 binds to receptor 20. In otherwords, the binding of analyte 12 and receptor 20 triggers aconformational change in sensor 10 resulting in a detectable change incharge transfer between first and second stems 14, 16. Theconformational change may consist of adaptive folding, compaction,structural stabilization or some other steric modification of junction18 in response to analyte binding which causes a change in the chargetransfer characteristics of sensor 10.

As shown in the embodiment of FIG. 1( a), receptor 20 is physicallycoupled to junction 18 but does not form a portion thereof. In thisembodiment receptor 20 is located proximate to first and second stems14, 16 but it does not form part of the conductive path in the secondconformational state. This is sometimes referred to herein as the“coupled receptor” or “coupled-ligand” embodiment. As shown in thealternative embodiment of FIG. 1( b), receptor 20 may form an integratedportion of junction 18 and hence part of the conductive path betweenfirst and second stems 14, 16 in the second conformational state (i.e.when analyte 12 binds to receptor 20). This is sometimes referred toherein as the “integrated receptor” or “integrated ligand” embodiment.

As shown in FIG. 1( a) sensor 10 may also optionally include a thirdoligonucleotide stem 22. Stem 22 may include receptor 20 and may bejoined to first and second stems 14, 16 at junction 18. In thisembodiment junction 18 therefore consists of a three-way junction.Sensor 10 may also comprise four-way or other multiple stem junctions asdescribed further below.

Sensor 10 further includes a charge flow inducer 24 for controllablyinducing charge transfer between first and second stems 14, 16 in thesecond conformational state. In one embodiment of the invention, thecharge flow inducer 24 may comprise a chemical trigger coupled to secondstem 16. For example, inducer 24 may consist of a photoexcitable,chemiexcitable or electrochemically excitable moiety which functions asa powerful oxidizing agent in its excited state. By way of example,suitable photoexcitable charge flow inducers 24 may include antraquinone(AQ) or rhodium (III) complexes with aromatic ligands (anthraquinone isshown as a charge flow inducer in illustrated embodiment). As will beappreciated by a person skilled in the art, inducer 24 couldalternatively comprise a powerful reducing agent configured to cause netcharge flow (i.e. in the direction opposite to that shown by the arrowin FIGS. 1( a) and 1(b) when triggered).

Charge transfer within sensor 10 may be detected either directly orindirectly in different embodiments of the invention. As shown in FIG.1( a), sensor 10 may include a detector 26 coupled to first stem 14 fordirectly detecting charge transfer between first and second stems 14, 16in the second conformational state. Detector 26 may comprise, forexample, a semi-conductor chip or some other conductive surface fordirect current measurements. In one alternative embodiment of theinvention, charge flow inducer 24 may be omitted if stems 14, 16 areeach directly connected to electrodes or other conductors (not shown)for directly inducing and measuring charge transfer between stems 14,16.

The use of DNA-based sensors 10 for direct electrical or electrochemicalmeasurements has many potential advantages in the field of gene chiptechnology. The technology for attaching DNA molecules to gold surfaceshas already been worked out and optimized³¹ Direct measurement ofdifferences in charge transfer may be employed to achieve rapid andautomated chip-based detection of small molecules as well as ofproteins, macromolecules and other analytes 12.

In another embodiment of the invention, charge transfer in the secondconformational state may be detected indirectly. For example, inducer 24may be selected to function, when triggered, as a powerful oxidizingagent causing the formation of oxidation products. As described indetail below, the position of oxidation products along a DNA strand maybe detected by sequencing gel-electrophoresis to indirectly detectcharge transfer. As mentioned above, one mechanism for explaining theelectrical conductivity of double-helical DNA is by means of a“multi-step hopping reaction” whereby guanine residues donate electronswhen subjected to oxidization. When triggered, inducer 24 acts as anelectron sink capable of collecting electrons from guanines viageneration of a mobile radical cation, or electron hole, within the DNAduplex. This effect has been reported at distances exceeding 200 Å awayfrom the oxidizing moiety^(15a,b)). According to the hopping reactionmechanism, the radical cation moves from guanine to guanine (guanine isthe base with the lowest ionization potential). A guanine upon which themobile radical cation is transiently localized is somewhat susceptibleto reaction with water and dissolved oxygen, leading to the formation ofoxidation products such as diaminooxazalone and 2-aminoimidazalone¹⁶.The position of the latter products along a DNA strand can readily bedetected by sequencing gel-electrophoresis, since these products arebase-labile and cause site-specific DNA strand cleavage when treatedwith hot piperidine. It has been noted, in particular, that stretches ofguanines, e.g. GG or GGG on a given strand, are especially susceptibleto oxidation, with the 5′-most guanine of these stretches usually themost oxidizable.

It is therefore believed that conduction through a regularly ordered DNAhelix (FIG. 2( a)) depends upon (a) the continuity of the π-stackinginteractions of the base pairs, and (b) on successive G-C base pairs inthe duplex being separated by no more than approximately 2 A-T basepairs. Other considerations apply in the case of more complexstructures, such as the sensors 10 of the present invention where, inthe first conformational state, base pairing is disrupted at junction 18between the first and second oligonucleotide stems 14, 16. For example,two unpaired nucleotides (i.e. non-Watson-Crick base pairs) may belocated at or near junction 18 (FIG. 2( b)). Guanine electron-donationmay be dependent upon the specific conformation of the three-wayjunction 18. In a standard three-way junction 18 (FIG. 2( c)) stericinterference between the three oligonucleotide stems 14, 16, 22 willnormally preclude ordered coaxial stacking of any two of the threestems. Therefore the passage of electrons from the guanines (shown asdark stubs in the drawings) of first stem 14 to moiety 26 connected tosecond stem 16 is less efficient relative to donation by the equivalentguanine in a conventionally ordered double helix. However, the presenceof two unpaired nucleotides at or near junction 18 (FIG. 2( a)) doesallow a 3-way junction to coaxially stack two of its three stems 14, 16,22. Variables affecting the efficiency of base pair stacking in theregion of junction 18 include the identity of the base pairs at thejunction itself as well as reaction conditions, such as the ionicconditions of the solution.

As indicated above, anthraquinone is convenient for use an charge flowinducer 24 since it is a chemically robust entity that is not easilydamaged by changes in temperature or pH. However, other such reagentsare known (including other organic moieties, as well as variousruthenium and rhodium organometallic complexes containing aromaticligands capable of intercalating into DNA), that are also suitable asphotooxidants.¹⁴ The ruthenium and rhodium complexes, moreover, usedtogether, can be used in a fluorescence-quenching detection method formonitoring charge conduction through DNA.

As will be apparent to a person skilled in the art, different means fordirectly and indirectly measuring changes in DNA conformation andconductivity are described in the literature and may be used inconjunction with the present invention. Fluorescence methods havetraditionally dominated instrumentation for DNA analysis. Thedisadvantages of fluorescent labeling are cost, linearity, sensitivityand the inherent need for analyte labeling. The detection of electricalproperties of DNA through charge transfer reactions is also known in theart. For example, Barton and co-workers have used electrocatalysis todetect single-base mismatches in double-stranded DNA immobilized on agold electrode surface by means of a thiol tether. Charge transportthrough the DNA duplex was detected as it shuttles from a redox-activeDNA intercalator (methylene blue) to a redox species (ferricyanide).²¹Direct measurement of current fluctuations on a gold or other metalsurface offers an attractive alternative to traditional fluorescencequenching.

The present invention could be adapted for use with other data readoutsystems for identifying changes in electrical conductivity caused byanalyte binding and for amplifying target signals to enhancesensitivity. For example, possible detection strategies includesurface-enhanced resonance Raman scattering, surface plasmon resonancemethods, acoustic wave sensors, and mass spectral analysis.

As illustrated in FIG. 3( a)-(d), sensor 10 may comprise a composite ofdifferent forms of nucleic acids in some embodiments of the invention(e.g. DNA, RNA and other modified or synthetic nucleotides). In thecell, DNA is found essentially in a double-helical form (except inspecialized elements, such as telomeres), whereas cellular RNAs arefound in a wide variety of complexly folded shapes. Consequently, too,there is a larger variety of modes of protein-RNA interaction found innature, relative to modes of DNA-protein interaction. In addition, invitro selection experiments have been carried out for a substantiallylarger number of RNA aptamers than DNA aptamers (although individual RNAand DNA aptamers are often of comparable quality, complexity, andligand-binding affinity)¹⁴. The large number of naturally occurringbinding sites and aptamers made of RNA may be exploited in the design ofsensors 10. For example, sensors 10 may be formed wholly from RNA, inwhich case charge conduction occurs through double-helical RNA. Inmodular sensors 10 a wholly DNA conduction path (i.e. oligonucleotides14, 16) is coupled to a wholly RNA receptor 20. In mixed sensors 10 theconducting path itself (i.e. oligonucleotides 14, 16) may comprise aRNA-DNA heteroduplex (FIGS. 3( a) and 3(b)).

As will be appreciated by a person skilled in the art, indirectdetection of charge transfer in oligonucleotide stems 14, 16 comprisingRNA double helices may require different protocols than double-strandedDNA. As described above, oxidation damage at specific guanines in DNA isgenerally observed by heating DNA with piperidine, a process that leadsto strand cleavage at the damaged guanines. With RNA, however, thisprocedure cannot be used, since RNA will be hydrolyzed non-specificallyby hot piperidine solutions. As an alternative, RNA double helices (inwhich one strand has been covalently derivatized at its 5′ end to acharge flow inducer 24, such as an anthraquinone moiety), may be treatedwith borohydride followed by hot aniline. This protocol is used togenerate a dimethylsulfate G-ladder²² for RNA, and it is expected thatit will work similarly to detect oxidation products of guanines derivedfrom RNA double helices. Of course, other direct or indirect chargetransfer detection means could be employed as discussed above in thecontext of DNA double helices.

Sensors 10 constructed from RNA stems 14, 16, 22 and receptors 20 may beuseful for the detection of specific types of RNA-binding analytes 12,such as HIV Rev proteins. The small HIV-coded proteins, Tat and Rev, areamong the best characterized of all RNA-binding proteins²³. Theirfunction is to bind to specific binding sites in the HIV genomic RNA (atthe TAR and RRE RNA loops), and the binding of both proteins is crucialfor the HIV life-cycle²³. In structural terms, the binding of Tat to TARand Rev to RRE, have the property of causing adaptive folding of theseRNA loops to compact and stacked structures. High resolution NMRstructures of both the TAR²⁴ and RRE²⁵ RNAs (with and without boundprotein) have indicated induced-fit folding of these RNA loops. Thebinding of Rev to RRE, in particular results in the closing of a large,and largely unstructured, bulge into a tightly hydrogen-bonded andstacked structure²⁵. This RRE loop will, therefore, lend itself toincorporation into an RNA sensor 10, for use as a receptor 20 of the HIVRev protein analyte 12.

The inventors have obtained a clone for HIV Rev, and purification ofthis protein is fairly simple. The RNA for constructing the sensors 10may be obtained using the in vitro T7 RNA polymerase system forunderivatized RNAs, and from chemical synthesis (where the RNA needs tobe derivatized with anthraquinone).

FIGS. 3( a) and 3(b) illustrate modular RNA sensors incorporatingRNA-DNA chimaeric stems 14, 16. Mixed sensors 10 comprising a simpleRNA/DNA heteroduplex are shown. A study on such heteroduplexes hasrecently been carried out by Barton²⁶, and it has been found that theydo indeed conduct charge similarly to DNA²⁶. The advantage of usingRNA-DNA heteroduplexes (relative to duplex RNA) is that the guaninesused for monitoring conduction can all be placed on the DNA strand and,hence, standard hot piperidine treatment can be carried out to detectoxidative damage to these guanines.

Depending upon the application, receptors 20 comprising RNA aptamers maybe more effective in binding a target analyte 12 than DNA aptamers.However, the sensitivity of detection may be dependent upon otherfactors as well, such as the “conformational information transmission”properties of the linking element (i.e. the structure or “communicationmodule” connecting receptor 20 to the 3-way junction 18 and to theconducting stems 14, 16).

As indicated above, sensors 20 may comprise a fourth or othersupplementary oligonucleotide stem 28 which is joined to stems 14, 16and 22 at a 4-way junction 18 (FIGS. 3( c) and 3(d)). The properties andconformational transitions of 4-way helical junctions 18 (immobileHolliday Junctions) have been studied extensively, using a wide varietyof techniques (reviewed in ref. 26 a), and their conformationalproperties are, on the whole, better understood than those of 3-wayjunctions 18. Unlike 3-way junctions, in which, depending on thecontext, two of the three (or none of the three) stems 14, 16, 22 maystack with each other (and, with different degrees of colinearity),4-way junctions 18 typically adopt a standard stacked geometry underphysiological salt conditions, with the four stems 14, 16, 22, 28stacking up in pairs to give an X-shaped structure (shown in twodimensions as an ‘H’ shape in FIGS. 3( c) and 3(d), right). The choiceof which 14, 16, 22, 28 stacks with which other is significantlydetermined by the identity of the base-pairs at the junction itself.Under low-salt conditions, however, 4-way junctions adopt an opened up,cross-shaped conformation (FIGS. 3( c) and 3(d), left) with minimalstacking between the four stems 14, 16, 22, 28.

In accordance with one embodiment of the invention, sensor 10 may beconfigured so that one of the four stems (e.g. stem 22 in FIGS. 3( c)and 3(d)) is a relatively unstructured receptor 20. According to thisdesign, junction 18, in the absence of added analyte 12, will adopt alooser structure than the tight X-structure of authentic 4-way junctions(i.e. resembling the looser structure shown in FIGS. 3( c) and 3(d),left). The binding of analyte 12 to receptor 20, however, should foldthat bulge into a helix-like stem; and, the presence, now, of thisadditional stem, should favour the formation of the classic X-shape. Insuch a design, one of the three preexisting stems of the junction wouldbe designated the first or detector stem 14—its identity empiricallydetermined as that stem most responsive—in terms of enhanced chargeconduction through its guanines—to binding of analyte 12.

Depending upon their configuration, 4-way junction-based sensors 10 mayprovide sharply differentiated yes/no responses to the presence ofanalyte (given the propensity of 4-way junctions 18 to exist in one oftwo states—stacked or unstacked). It is also possible that bettersignal-to-noise ratios may be achieved with 4-way junction sensors 10than with their 3-way junction counterparts. As will be appreciated by aperson skilled in the art, five-way or higher number junctions 18 may beemployed in embodiments of the invention where more complex switchingfunctionalities are desirable.

As shown in FIGS. 4( a) and 4(b), sensors 10 may be specificallyconfigured to detect protein analytes 12. In one mode, binding of theprotein analyte 12 to receptor 20 physically inteferes with theelectronic path between stems 14, 16 (and hence is detectable asreduction in charge transfer). In a second mode, the protein analytebinds to receptor 20, which may comprise a complexly structured DNA orRNA binding site, and cause an adaptive tightening and stabilization ofthat binding site (which, in turn, can be transmitted to stabilize orimprove the charge conduction path).

FIG. 4( a) illustrates the binding of a relatively large protein analyte12 to either its natural binding site (for a DNA-binding protein) or toan aptamer element (for a non-DNA-binding protein) located at receptor20 on stem 22. In this embodiment receptor 20 is located proximate to3-way junction 18. Binding of the protein analyte 12 to receptor 20could, by a process of physical interference by the protein (if theprotein were bulky enough and positioned correctly with respect to the3-way junction 18), alter the stacking geometry and, hence, theconduction path between stems 14, 16. Naturally DNA-binding proteins,including transcription factors such as the TATA-binding protein,glucocorticoid receptor proteins, GAL4 from yeast, or CAP or the lacrepressor from E. coli could be suitable, for example, for this sort ofdetection. As will be appreciated to a person skilled in the art, thespecific location and orientation of receptor 20 on stem 22 (such as itsdistance from junction 18 and its position along the helical path of thestem) could be selected to ensure a steric clash of the bound proteinwith the first and second stems 14, 16.

The sensor design shown in FIG. 4( a) is configured to detect thebinding of the cognate protein analyte 12 by a net decrease of chargeflow through stems 14, 16. Alternatively sensor 10 could be configuredto yield an increase in current upon analyte binding. This maynecessitate starting out with a different design of 3-way junction 18,perhaps such that the second stem 16 is initially stacked with the thirdstem 22 (containing receptor 20) rather than the first stem 14.

Another potential way to detect the binding of certain protein analytes12 (ideally, smaller proteins) might be on the basis of the adaptivefolding of their RNA and DNA binding site(s) on receptor 20 (FIG. 4(b)). In exhibiting such binding behaviour, these proteins wouldfunctionally resemble the small molecule analytes 12 (discussed above).

FIG. 5 illustrates an embodiment of the invention for use in binding anddetection of nucleic acid analytes 12 (i.e. DNA or RNA oligomers). Inthis embodiment incomplete 3-way junctions 18 will be constructed, whichwill incorporate two complete stems 14, 16, coaxially stacked, as wellas one of the two strands of a potential third stem 22. This danglingsingle-strand stem 22 would, essentially function as a receptor 20 forits complementary nucleotide sequence. In principle, the binding of anoligonucleotide of the correct length and with the correctcomplementarity to the dangling single-strand stem 22 (under definedconditions of hybridization) would complete a strained 3-way junction 18(FIG. 5). In the illustrated embodiment, this results in theintroduction of an enhanced steric strain in junction 18. The poorstacking of helical stems 14, 16 in such a strained junction 18 (i.e. inthe second conformational state) would lead to poorer conduction infirst stem 14 relative to its more orderly stacked precursor (in thefirst conformational state). The single-stranded element comprisingreceptor 20, 15-18 nucleotides long, could in principle have anysequence or length, although the illustrated embodiment is on the orderof 15-18 nucleotides long. Receptor 20 could even represent a collectionof sequences to make up a library sufficient to specify every15-18-nucleotide stretch of sequence present within a ˜10⁹ base pairgenome.

The present invention also has potential application as a DNA conductorused in gene chips. Such chips typically consist of arrays ofsingle-stranded oligomers, which are poor conductors. When, however, acomplementary strand hybridizes to a given oligomer on the chip, thatnewly-formed double helix can be detected by virtue of its superiorconductivity. This is an interesting conception; however theconductivity of individual double helices formed in this way will varywidely (depending on the guanine-content of the duplex, and the locationof those guanines relative to one another). In other words, theelectrical signal measured will vary, depending on what duplex has beenproduced, and this may give rise to ambiguity as to whether a positive(i.e. hybridization) signal is being observed or not.

One advantage of Applicant's invention as described above is that thetwo stacked stems 14, 16 making up the charge flow path could remainconstant for the entire library and hence a clear signal will begenerated for every single case of hybridization of a target analyte 12to a receptor 20, for example located on a third stem 22. Thus, ahybridization event to a receptor 20 of any sequence should produce astandard conductivity enhancement (or decrease, as the case may be) inthe conduction path as the sensor switches between the first and secondconformational states.

As will be apparent to a person skilled in the art, various enhancementsand modifications to the DNA/RNA conductor backbone and switchingconfigurations of sensors 10 are possible without departing from theinvention. For example, as indicated above, the structure of thenucleotide stems 14, 16, 22 and aptamer sequences at receptor site 20may be varied to include non-naturally occurring molecules, includingnucleic acid analogues and nucleic acid-like molecules capable of basepairing. The synthesis and properties of modified oligonucleotides hasbeen described in the literature (e.g. Modifiedoligonucleotides-synthesis, properties and applications, Iyer et al.,Current Opinion in Molecular Therapeutics (1999) 1(3):344-358; BackboneModification of Nucleic Acids: Synthesis, Structure and TherapeuticApplications, Micklefield, Current Medicinal Chemistry, 2001, 8,1157-1159, the text of which is incorporated herein by reference).Synthetic oligonucleotides could be configured, for example, to increasestability to enzymatic degradation, enhance affinity for binding totarget analytes 12 or to optimize electrical conductivity.

Sensors 10 of the present invention are also potentially useful asnanoelectronic switches and junction devices simulating solid stateelectronic logic gates. As shown in FIG. 6-9, each DNA sensor 10 isoperable in one of two distinct states (e.g. “on” or “off” or“conducting” or “non-conducting”). FIG. 6 shows an “AND” logic gate. Asin several of the other embodiments described, a charge flow inducer 24,such as the oxidant anthraquinone “AQ”, is tethered covalently to theend of a second stem 16. A pair of receptors 20(a) and 20(b) aredisposed between the first and second stems 14, 16. In order to achieveelectron flow in this embodiment between stems 14, 16, both of analytes12(a) and 12(b) must bind to their respective receptors 20(a) and 20(b).That is, binding of only one analyte 12(a), 12(b) is not sufficient totrigger electron flow between stems 14, 16.

FIG. 7 shows a “NAND” logic gate. In the “NAND” gate embodimentreceptors 20(a) and 20(b) are configured to ordinarily not interruptelectron flow between the first and second stems 14, 16. However, whenboth of analytes 12(a) and 12(b) bind to their respective receptors,stems 14, 16 undergo a conformational change resulting in cessation or areduction in electron flow (e.g. sensor 10 switches from the first tothe second conformational state). As will be appreciated by a personskilled in the art, other logic gate configurations employing coupledreceptor sensors 20 could be chosen, such as “OR”, “NOT”, depending uponthe functionality required.

FIGS. 8-9 illustrate a similar concept employing a single coupledreceptor sensor 10 disposed at junction 18 between first, second andthird stems 14, 16 and 22. In Figure the gate is ordinarily “ON” but maybe switched “OFF” upon the binding of analyte 12 which alters theconformation of at least some of stems 14, 16, 22 at three-way junction18. The opposite configuration is shown in FIG. 9. That is, the logicgate is ordinarily “OFF” but may be switched “ON” upon the binding ofanalyte 12. These examples highlight the versatility and scalability ofthe coupled receptor sensor/switch concept described herein (i.e. whereanalyte binding event(s) are transformed into electrical signal(s)).

FIG. 10 illustrates an in vitro selection protocol approach forrationally designing sensors 10 for different categories of smallmolecules and macromolecules or other analytes 12. The protocol may bepotentially employed to select receptors 20 for any molecule (regardlessof whether there exists any natural RNA/DNA binding site for such amolecule or, indeed, an aptamer).

FIG. 10 shows the design of a random sequence-containing library of10¹⁴⁻¹⁵ individual sequences, and a scheme for the selection ofreceptors 20, to be carried out in the solution phase (as opposed tobeing immobilized on a column). A random sequence duplex DNA library(FIG. 10, top), containing an N₄₀ random sequence region as well as asingle 8-oxoguanine (⁸ _(G)) residue (a highly oxidizable baseanalogue²⁷, superior to guanine, but too expensive to use routinely) inone of the two strands, will be treated in a standard fashion to obtainthe ⁸ _(G)-containing strand as a single strand (the protocol for doingthis involves immobilizing the duplex onto an avidin column using asingle biotin attached to the non-⁸ _(G)-containing strand, and theneluting off the other strand with a 0.2N sodium hydroxide solution). Thesingle-stranded library so obtained will be hybridized to afixed-sequence, partially complementary, AQ strand (i.e. the second stem16), containing a covalently attached anthraquinone residue on its 5′end. The hybridization of the library strand with the second stem 16should give rise to a partial heteroduplex (FIG. 10, right), in whichthe N₄₀ element is looped out. It is anticipated, based on priorexperience in the field¹⁰, that aptamer- or receptor binding-elementsfor particular analytes will emerge out of this N₄₀ element during thecycles of selection.

The following, then, are the detailed features of this starting DNAcomplex (FIG. 10, right) for our selections: the AQ strand, 45nucleotides long, and of predetermined sequence, will have at its 5′ enda covalently tethered anthraquinone (AQ) moiety. The other strand (the“random strand”), 80 nucleotides long, will have, as its 5′ and 3′extremities, a 15-nucleotide and a 25-nucleotide fixed sequencestretches, fully complementary to the AQ strand. In the remaining 40nucleotides of this random strand (N₄₀) each nucleotide position willhave an equal probability of being an A, G, C, or T (the generation ofsuch random-sequence stretches within synthetic DNA molecules isroutinely achieved by automated synthesis). The “random” strand willalso be ³²P-labelled at its 5′ end, and will feature the single8-oxoguanine residue, located 10 nucleotides away from the 5′ end.

FIG. 10 shows how the binding of a chosen analyte (i.e. ligand) to oneor more individual sequences within the N₄₀ element (that mightconstitute a binding site, or aptamer, for it)—may lead to an adaptivefolding of this binding sequence, in turn leading to an aligning andstacking of the first and second stems 14, 16, enhancing the currentflow through them (with enhanced oxidative damage to the 8-oxoguanineresidue). In other words, the tight binding of analyte to one or moreindividual members of the ‘random’ DNA constructs, might, at least in aproportion of cases, lead to enchanced oxidative damage to the8-oxoguanine residue within those constructs. Heating with piperidinewill then break these oxidized strands, shortening them by 10nucleotides relative to unoxidized strands. These shortened strands caneasily be separated and purified by gel electrophoresis, recovered, andre-converted, using PCR, to full-length random strands to be used forthe next round of selection.

In carrying out such a selection we will have to take certain keyprecautions: (a) the unirradiated “random strand” library will need tobe pre-heated (prior to the first round of selection) with piperidine,to eliminate DNA strands that have collected lesions during automatedDNA synthesis. Such a pre-treatment with piperidine, in the absence ofprior oxidation, should not harm the 8-oxoguanine residue, which isrelatively insensitive to piperidine. However, if this pre-treatmentleads to a high ‘background’ cleavage at the 8-oxoguanine, we can switchto GG or GGG sequences to replace the 8-oxoguanine in our librarydesign. (b) Stringent negative selections (i.e. in the absence of addedanalyte) will be carried out, to element random sequences thatcontribute to enhanced current flow along the first and second stemseven in the absence of added analyte.

A feature of the design shown in FIG. 10 is the presence of fourostensibly unpaired nucleotides (GCTA) at the centre of the second stem16. These will permit a degree of flexibility in the choice of junctionbase pairs by emerging 3-way junction sensors during selection.

FIG. 11( b) illustrates an embodiment of the invention in which a sensorhas been coupled to a chip for the detection of an analyte. A sensor 10is coupled to a chip 30 for the direct detection of an analyte 12.Detector stem 14′ is coupled to chip 30 to anchor sensor 10 to chip 30.Sensor 10 may be coupled to chip 30 by any means known to those skilledin the art. For example, chip 30 may be a gold chip, in which casesensor 10 may include a thiol group, so that a plurality of sensors 10may be coupled to gold chip 30 via a sulfur-gold linkage to form arobust DNA monolayer on the surface of chip 30. There are otheralternative substrate materials, such as Pt, C, or semiconductors (Si,ITO), for which cases the DNA/RNA sensors may be attached by differentsurface reactions. For example, on silicon surfaces, acarboxyl-terminated linker monolayer will be formed first, NH₂-modifiedDNA strands will be coupled to the surface accordingly. The open surfaceof chip 30 may be passivated with inert alkanethiols, to reducenonspecific adsorption and current leakage. For example,6-mercaptohexanol (MCH) may be used to passivate the open surface ofchip 30 to prevent non-specific adsorption and to reduce leakagecurrents. Alkanethiols with different chain lengths or functional groupscould be used to accomplish this task.

A suitable redox indicator 34 may be coupled to reporter stem 16′ topermit detection of an analyte using protocols that are known to thoseskilled in the art.⁵¹

As an example, ferrocene (Fc) may be covalently attached to the free endof reporter stem 16′ to allow for electrochemical monitoring of thebinding event. If utilized, Fc may be pre-oxidized to the form Fc⁺ inorder to accept an electron, rather than being used as a reducing agentto release an electron. Other redox indicators, such as methylene blueor transition metal complexes, may also be used to permitelectrochemical monitoring of a binding event. Binding of analyte 12 toreceptor 20 will permit the flow of electrons, either from the chip 30towards the redox indicator 34, or from the redox indicator 34 towardsthe chip 30, thereby producing an electrical current which may bemeasured. Thus, a chip-bound sensor according to this embodiment permitsthe rapid detection of analyte 12 in solution.

One particular application where the present invention could be employedis in the screening of compounds potentially useful as drugs. Forexample, the sensors could be configured to detect the presence ofenzymes known to be associated with a particular disease state. Thesensors could be deployed on a chip for automated chip-based detectionand quantification of the enzymes or other small molecule analytes asdescribed herein. A drug candidate could then be introduced into thetest system to determine whether its presence results in any change inelectrical conductivity. More particularly, the binding of adisease-related enzyme to its cognate sensor could result in a definingcharge flow through the sensor. Then, the binding of a metabolite, smallmolecule or protein to the sensor-bound enzyme would result in a furtherdefining change in the charge flow characteristics of the sensor. Thechange in charge flow characteristics could, for example, bespecifically correlated with a change in conformation of thesensor-bound enzyme suggestive of an inhibitory effect. As will beappreciated by a person skilled in the art, many other potential uses ofthe sensors in directly or indirectly identifying drug candidates arepossible.

EXAMPLES 2.1 Example Synopsis

FIGS. 12-16 illustrate an illustrative embodiment of the invention fordetecting the presence of the analyte adenosine, which binds poorly, ifat all, to double-stranded DNA but for which a high-affinity (K_(d)˜μM)DNA aptamer sequence has been derived²⁸. NMR studies have confirmed thatthis aptamer, upon binding two molecules of adenosine, shows a typicaladaptive folding, forming a tightly hydrogen-bonded and stacked helicalstructure²⁹. Accordingly, in this example, analyte 12 is adenosine andreceptor 20 is the adenosine aptamer sequence. Antraquinone is used as acharge flow inducer 24, namely a photoexcitable moiety covalentlycoupled to second stem 16 for controllably inducing charge transfer.

2.2 Materials and Methods 2.2.1 DNA Preparation.

Umnodified DNA sequences were purchased from Sigma-Genosys and purifiedby PAGE before use. Sequences to be ³²P-end-labeled were pre-treatedwith 10% piperidine (90° C. for 30 minutes followed by lyophilization)prior to 5′-³²P-kinasing and PAGE purification. Such a pre-treatmentcleaved DNA molecules damaged during synthesis, leading in turn to lowerbackground cleavages from photo-irradiation experiments, as previouslydescribed³⁰.

DNA sequences to be derivatized with anthraquinone were synthesized witha commercially available 5′-C6-amino functionality, and were purchasedfrom the University of Calgary Core DNA Services. Generation ofanthraquinone-modified DNA sequences was accomplished by reacting theN-hydroxysuccinimide ester of anthraquinone-2-carboxylic acid³¹ with the5′-C6-amino functionality on the DNA. Coupling and purificationprotocols were as described for amine reactive dyes by MolecularProbes³² with some modifications.

Prior to coupling, the DNA was treated to remove nitrogenouscontaminants from the DNA synthesis procedures. The dried DNA sampleswere first suspended in 100 μl ddH2O, and were extracted three timeswith 100 μl of chloroform. The DNA remaining in the aqueous phase wasthen precipitated by the addition of 30 μl 1M NaCl and 340 μl 100% EtOH.Following mixing, the sample was chilled on dry ice for ˜10 minutes, andthen centrifuged in a microfuge for 20 minutes to pellet the DNA. Thepellet was washed once with 150 μl of 70% aqueous ethanol (v/v).Following air-drying the pellet was dissolved in 100 μl ddH₂O, the DNAconcentration of the solution was determined in a standard fashion usingUV absorbance measurements.

The AQ-NHS ester (4.8 mg) was dissolved in 238 μl dimethylformamide. Foreach coupling reaction, 7 μl of this stock suspension was added to 75 μlof a 100 mM sodium borate solution (pH 8.5). To the resulting mixturewas added 8-15 μl (5-10 nmoles) of the purified amino-labeled DNA. Thetubes containing the coupling mixtures were covered in aluminum foil andshaken overnight at room temperature. The DNA was then ethanolprecipitated by addition of 27 μl of 1M NaCl and 280 μl of 100% ethanol(the solution was chilled in dry ice and the precipitated DNA collectedand washed as described above). The large pellet obtained (containing asignificant amount of the uncoupled anthraquinone) was now suspended in50 μl of 100 mM aqueous triethylamine acetate (pH 6.9), to which wasadded 100 μl chloroform. The uncoupled anthraquinone partititioned intothe chloroform phase, and the aqueous phase was now extracted two moretimes with 100 μl chloroform, prior to partial drying under vacuum toremove any residual chloroform. The DNA obtained was then purified byreverse phase chromatography on an HPLC using a C18 Bondapack column(Waters).

The HPLC protocol was as follows. The solvent flow was continuous at 1ml/minute, and the column was heated to 65° C. The initial conditionswere: 100% Solvent A (20:1 of 100 mM triethylamine acetate, pH 6.9:acetonitrile) changing to 30% Solvent B (100% acetonitrile), over 30minutes and with a linear gradient. After this period, the solvent wasrapidly changed to 100% Solvent B, for 15 minutes, before reconditioningthe column to the starting conditions.

The concentrations of the various products of the coupling reactionscould be determined spectroscopically. Absorbance values for theconjugate were made at 260 nm, using extinction coefficients for theindividual bases obtained for single stranded DNA. ε (260 nm, M⁻¹ cm⁻¹):adenine (A)=15,000; guanine (G)=12,300; cytosine (C)=7,400; thymine(T)=6,700; and, anthraquinone (AQ)=29,000. Typical yields of AQ-DNAconjugates ranged from 50-85% depending on the sequence of the DNAoligonucleotide being coupled, and the synthetic batch.

2.2.2 Preparation of DNA Assemblies.

DNA assemblies were formed by annealing mixtures of constituent DNAoligonucleotides (1 μM each) in 100 mM Tris-Cl, pH 7.9, and 0.2 mM EDTA.DNA solutions were heated to 90° C. for 2 minutes, and then cooled at arate of 2° C./minute to a final temperature of 20° C. The solutions werethen diluted two-fold with either 5 nM MgCl₂, or with 5 mM MgCl₂ and 200mM NaCl (the final solutions being defined as the ‘Mg’ and ‘Mg—Na’buffers, respectively). These solutions also contained 2× concentrationsof adenosine or uridine in some samples. After mixing, the samples wereincubated for approximately 30 minutes at room temperature beforephoto-irradiation.

2.2.3 Photo-Irradiation.

Pre-incubated samples were placed under a UVP Black-Ray UVL-56 lamp (366nm peak intensity, at 18 W) for 90 minutes at a distance of 4 cm fromthe bulb. Temperature was maintained by having the samples tubes placedin a water bath set to the desired temperature. Followingphoto-irradiation, the samples were lyophilized, and then treated withhot piperidine as described above. The treated DNA samples were thenloaded on 11-12% sequencing gels and analyzed using a BioRadPhosophorimager.

2.3 Results 2.3.1 The Integrated-Ligand Sensor.

Experimentally, the most sensitive way to monitor changes in electricalconductivity at the level of individual nucleotides is toelectrophoretically monitor DNA strand cleavage resulting frombase-labile oxidative damage suffered by individual guanines³³. Thismethod of monitoring charge transport, often termed “water trapping”,has been successfully used to monitor the long-range effects (>150Å)^(34,35a) of a variety of oxidant groups covalently attached atdefined sites on the DNA (reviewed by Grinstaff^(35b)). FIG. 12 showsthe DNA sequences and schematic of a potential “integrated” adenosinesensor, and of a “control” Watson-Crick duplex construct (the³²P-labeled strand is identical in the two constructs). The 5′³²P-labeled strands in the constructs contain guanine doublets (GG) oneither side of the ATP aptamer domain (or its Watson-Crick base-pairedcounterpart). The proximal (“P”) guanine doublet allows convenientmonitoring of charge transfer in the second (i.e. AQ stem) 16, while thedistal (“D”) doublet permits the same for the first (i.e. detector) stem14. Guanine doublets have been used in preference to isolated singleguanines for charge transfer measurements because of the former's higherreactivity (particularly that of the 5′ guanine) in water trappingexperiments³⁶. The particular base composition of the duplex immediatelyadjacent to the AQ-tether was chosen because this sequence has beenshown to promote efficient charge transfer from the tethered andphoto-excited anthraquinone³⁷.

Upon photo-irradiation at 366 nm wavelength in Mg—Na buffer (50 mMTris-Cl, pH 7.9, 2.5 mM MgCl₂, 100 mM NaCl and 0.1 mM EDTA), theanthraquinone-modified DNA constructs shown in FIG. 13 showeddistinctive oxidative cleavage patterns. Both the control duplex and theintegrated-ligand sensor 10 exhibited significant levels of strandcleavage at the proximal (“P”) guanine doublet in the presence of 2.5 mMadenosine (FIG. 14( a), lanes 5 and 12), 2.5 mM uridine (lanes 6 and 13)or no added nucleoside (lanes 4 and 11), relative to the ‘dark’ (i.e.unirradiated—lanes 3 and 10) controls. Dramatic differences between theconstructs, however, were observed in cleavage at the distal (“D”)guanine doublet. In both the presence and absence of added nucleosidesthe control duplex exhibited identical levels of cleavage at the “D”guanines (lanes 4-6). However, the integrated-ligand sensor onlyexhibited significant cleavage at the “D” guanines in the presence ofadenosine (lane 12), but not in the presence of uridine (lane 13), noadded nucleoside (lane 11), or, in a ‘dark’ (i.e. unirradiated—lane 10)control. To test whether the cleavages observed arose uniquely fromoxidation by the attached anthraquinone functionality, and also to testwhether such putatively AQ-dependent cleavages occurred strictly inintra-molecular fashion, photo-irradiation was carried out on samplescontaining mixtures of ³²P-end-labeled constructs lacking anthraquinoneand unlabeled constructs conjugated to anthraquinone. Lanes 7 and 14show that no significant cleavage in the labeled strands was observed,either for the double-stranded control or the integrated-ligand sensorconstruct.

The fact that in the integrated-ligand sensor high levels of strandcleavage were observed only at the proximal (“P”) guanine doublet in thepresence of uridine (lane 13), as well as in the absence of addednucleosides (lane 11, FIG. 14( a)), indicated that charge transport inthese cases was localized almost exclusively within the AQ stem 16 (asdepicted in the model for this sensor 10 in FIG. 12). By contrast, when2.5 mM adenosine was present (lane 12) the same experimental procedureresulted in 5.0% cleavage at the distal (“D”) guanine doublet (up from0.26% shown in lanes 11 and 13) when photo-irradiated for 90 minutes.This reflects a 20 fold enhancement in strand-cleavage enhancement inthe presence of the adenosine ligand analyte 12. This observationindicates that the adenosine-induced folded structure of the aptamerreceptor 20 was indeed capable of facilitating charge transfer betweenthe first (detector) and second (AQ) stems 14, 16. Interestingly, in thepresence of adenosine, even the “P” doublet showed a small enhancementin cleavage (<2 fold), consistent with an overall tightening andstabilization of the sensor construct.

When experiments similar to the above were carried out in the absence ofNaCl (i.e. in the ‘Mg’ buffer: 50 mM Tris-Cl, pH 7.9, 2.5 mM MgCl₂, and0.1 mM EDTA), somewhat different results were observed (FIG. 14( b)).The double-stranded control showed comparable cleavage patterns as seenin the ‘Mg—Na’ buffer, i.e. strand cleavage was observed at the proximal(P) and distal (D) guanine doublets both in the absence (lane 4) orpresence (data not shown) of 2 mM adenosine (relative to the ‘dark’control—lane 3). By contrast, the integrated sensor construct exhibitedsignificant strand cleavage at the distal (D) guanine doublet in thepresence of 2 mM added adenosine (lane 12)—as also seen in the ‘Mg—Na’buffer (FIG. 14( a)). This increase in strand cleavage, from 0.98% to7.3%, describes a 7 fold enhancement in the presence of adenosine.

The major difference observed for the sensor construct in the ‘Mg’buffer (FIG. 14( b)), relative to the ‘Mg—Na’ buffer (FIG. 14( a)), wasthat in the ‘Mg’ buffer enhanced cleavage occurred at the proximal (P)guanine doublet in the presence of adenosine (lane 12 versus lanes 11and 13, FIG. 14( b)). This enhanced cleavage may reflect aproportionately greater stabilization of the duplex elements flankingthe aptamer receptor 20 by the aptamer-bound adenosines in thisrelatively low-salt buffer. The inventors believe that this enhancedproximal G cleavage was not a consequence of the re-association of thestrands of the sensor construct dissociated in the ‘Mg’ buffer(non-denaturing gel electrophoresis experiments showed that theconstructs remained intact in all buffers and experimental conditionsused in this study—data not shown).

Variations in solution conditions also affected the efficiency of strandcleavage at the distal (D) guanine doublet as a function of adenosineconcentration. FIG. 15 shows how in different solutions differentbinding affinities were observed for the adenosine ligand. In the ‘Mg’buffer, half maximal strand cleavage was observed at 18 μM adenosine,while in the ‘Mg—Na’ buffer, it was observed at 135 μM adenosine.

2.3.2 The Coupled-Ligand Sensor.

The properties of a different sensor design, the “coupled-ligand” sensor(which also utilized the ATP aptamer, and is shown schematically in FIG.12, lower) were also examined. This second design does not depend on theconductive property of the folded aptamer domain. The predicted lack ofbase stacking between the third stem (i.e. sensor) 22 stem and either ofthe first and second stems 14, 16 in the folded state was expected toprevent electron transfer between these regions. A coupled-ligand sensor10 of this design, for detecting adenosine, was assembled from the DNAoligomers shown in FIG. 16( a). In this construct, the aptamer bulge wasseparated from the three-way junction 18 by a single A-T base pair. Todetect charge transfer in the various stems, the DNA strand shared byboth the first and third stems 14, 22 was 5′-³²P-labelled. FIG. 16( b)shows the results obtained when samples were irradiated in the ‘Mg—Na’buffer for 180 minutes. In the absence of added adenosine (lane 5), orin the presence of 2.5 mM uridine, no detectable cleavage above thebackground level (lane 6) was observed at all positions. In the presenceof 2.5 mM adenosine (lane 4), however, significantly enhanced strandcleavage (>15 fold enhancement in replicate experiments) was seen at the5′ guanine of each of the two-guanine doublets present in the first stem14 (indicated as x and y). Lack of detectable cleavage at these guaninesin the absence of adenosine prevented the determination of an absoluteratio for cleavage enhancement; however, the lower limit indicated above(>15 fold) could be calculated. A comparable enhancement, however, wasnot observed for the doublet (z) located in the third stem 22 (2-4 foldincrease) as predicted by a structural model of this DNA construct.Irradiation experiments on a control construct for the above three-wayjunction 18, that lacked the aptamer bulge but incorporated aWatson-Crick duplex (as with the ‘integrated sensor’—see above) in placeof the aptamer ‘arm’, yielded no modulation of strand cleavage in thepresence of added adenosine (data not shown).

The effect of different buffer conditions on strand cleavage in thisconstruct was not examined, as non-denaturing electrophoresisexperiments indicated that this sensor construct was not sufficientlystable structurally in the low-salt ‘Mg’ buffer (data not shown).

2.4 Discussion

Experiments with the above two sensor designs, utilizing the ATP aptameras a receptor 20, clearly demonstrate the utility of DNA conformationalchanges (resulting from the adaptive binding of analyte 12 to the DNAaptamer) in modulating charge-transfer through DNA.

Investigations with the integrated-ligand sensor 10; above, havedemonstrated that both the sensitivity of and signal enhancement fromanalyte-sensing depend significantly on solution conditions. It remainsunclear whether such differences reflect purely structuraltransformations of the aptamer in the different ionic strength solutionsor whether they also reflect changes in the process of charge-transferthrough DNA.

Comparison of the behavior of the integrated sensor 10 in the two ionicstrength conditions tested indicates a trade-off between signalenhancement and sensitivity. In the relatively low-salt ‘Mg’ buffer ahalf maximal enhancement of ˜3.5 fold enhanced cleavage at the distal(“D”) guanine doublet was observed with 18 μM adenosine, whereas in thehigher salt ‘Mg—Na’ buffer a half maximal enhancement of ˜10 fold wasobserved with 130 μM adenosine. In other words, the addition of 100 mMNaCl generated a highly amplified signal, but at the cost of a lowersensitivity of adenosine detection. Such an unusual trend may resultfrom the aptamer forming a subtly altered structure under higher ionicstrength conditions, one that requires higher adenosine concentrationsto drive the equilibrium to the adenosine-bound form. The guanine richaptamer domain could potentially form foldback G-G base pairs or guaninequartets in the presence of NaCl³⁸⁻³⁹. In fact, guanine quartets wereoriginally postulated to be a part of the folded aptamer structure whenthe aptamer was originally identified²⁸.

In addition, care must be taken in interpreting the results of theadenosine dependence data from FIG. 15, since the curves may notdirectly reflect the binding affinities of the aptamer for its ligand.As described above, each molecule of this particular aptamer binds twomolecules of the adenosine ligand, and it is unclear whether the bindingof only one molecule of ligand allows charge transfer to occur to someextent or not.

Comparing the efficiencies of conduction through particular DNAsequences has often been accomplished by comparing the ratios ofcleavage at “proximal” and “distal” guanines (D/P or P/Dratios)^(7,8,34,26,40,41). This comparison is an indicator of theefficiency with which charge transfer proceeds through a specifiedsequence of interest that is flanked by isolated guanines, doublets,triplets, or reactive bases such as 8-oxoguanine⁹ or 7-deazaguanine⁴¹. Acomparison of conduction through the integrated sensor and through itsdouble-stranded control gave D/P ratios, respectively, of 0.48±0.07versus 0.23±0.04 in the ‘Mg—Na’ buffer; and, 0.4±0.08 versus 0.24±0.05in the ‘Mg’ buffer. Comparing these ratios at face value suggests thatthe aptamer domain is a somewhat superior conductor compared to thedouble-stranded DNA control. However, direct comparisons of the sensor10 and the duplex control may not be entirely appropriate. Whenirradiation experiments were carried out such that comparable levels ofstrand cleavage were achieved at the distal (“D”) guanine doublets inboth the control and sensor constructs, a two-fold higher cleavage wasobserved at the proximal (“P”) guanine doublet of the double strandedcontrol. The reduced efficiency of cleavage at the proximal guanines inthe sensor construct must reflect a hindrance to charge migration intosequences influenced by the presence of the aptamer, given that thedouble stranded proximal stems are identical in both the sensor anddouble stranded constructs. Once a mobile charge reaches the proximal(“P”) guanine doublet, it then appears that the ‘integrated-ligand’sensor better facilitates the transfer of that charge to the distal(“D”) guanine doublet in comparison to the double stranded control. Apossible reason for the lower D/P ratios seen in the duplex control mayresult from the GG doublets situated between the “P” and “D” doublets onthe AQ-modified strand (FIG. 13). These intervening doublets may beacting as ‘traps’, thus reducing the efficiency of charge transfer tothe distal (“D”) guanine doublet of the duplex control. Overall, it isstill remarkable that the folded, non-B-DNA aptamer possesses acomparable if not more favorable conductive property than the B-DNAduplex control.

Besides the capacity of the ATP aptamer to modulate charge transferbetween the acceptor and detector stems, interesting observations weremade regarding the aptamer domain, specifically. Cleavage at theguanines located within the aptamer domain was observed to be low inboth of the buffers used (FIG. 14( a) & 14(b), lanes 11-13). Lowcleavage in the absence of bound adenosine was lowered further upon thebinding of adenosine. This observation may reflect the non-B helicalstructure of the folded aptamer domain. The high level of oxidation ofthe 5′-most guanine in guanine doublets is strictly true only fordouble-stranded B-DNA⁴². Single-stranded sequences¹⁰, and guaninequadruplexes⁴³, for instance, do not show this pattern. This property ofthe aptamer guanines may explain the lower D/P ratio of the sensor,relative to that of the duplex control.

The demonstrated capacity of the DNA aptamer for adenosine/ATP to act asa conduit for charge transfer in the folded state is a property notlikely shared by all aptamer motifs. In addition to inherentconductivity differences between different aptamers, some aptamers,which are not formed from internal (bulge) loops, may not easily beincorporated into duplex DNA. To design a more general sensor, capableof utilizing diverse receptors and aptamers, and responding to a varietyof ligands/analytes, an immobile three-way helical junction was used asa starting point for a second design. This ‘coupled-ligand’ sensor (FIG.16( a)) also exhibited modulation of charge transfer from the Acceptorto Detector stems in response to adenosines binding to the aptamerelement. This construct, however, required longer irradiation times(relative to that required for the integrated sensor) to obtainsignificant levels of cleavage at the guanine doublets (x and y in FIG.16( a)) in the Detector stem. It remains to be investigated whether thelower charge transfer efficiency in the ‘coupled-ligand’ construct arosefrom the particular sequences chosen for the stems, or from the presenceof the 3-way junction. Future work will also focus on determining thethree-dimensional structure of this 3-way junction, in order tounderstand why significantly more strand cleavage was observed in theDetector stem compared to the Sensor stem.

A deeper characterization of the ‘coupled-ligand’ sensor design, and ofrelated architectures, is desirable given their broad potential fordevelopment as modular sensors. In the ‘coupled-ligand’ sensor there isonly a requirement for a conformational change in the analyte-binding(aptamer or receptor) domain upon the binding of the analyte, and notfor an inherent ability of the binding domain to permit charge transferthrough its own structure. The side-on placement of the analyte receptorshould also be applicable in the design of hybrid sensors that are notcomposed entirely of DNA. Such hybrid systems may possess bindingdomains consisting of RNA, proteins or other organic “host” entities(such as crown ethers, cryptands, and others) that undergo aconformational change upon binding the appropriate “guest” molecule orion.

The guanine damage- and electrophoresis-based detection methodology usedin this Example were necessary for single-nucleotide resolutioninvestigations of the charge conduction pathways in the sensors. Here,we have used them to demonstrate that ligand-induced conformationalchanges can indeed be used to modulate charge transfer through DNA.Other detection methods, such as having the DNA sensor constructsfunctionalized onto metal or other surfaces such that directmeasurements of current flow can be made may be used, as discussedbelow. Reports of successful coupling of modified DNA to electrodes andthe direct monitoring (by chemical reaction in solution orphoto-excitation) of hybridization via charge transfer through theresulting duplex⁵,⁴⁴⁻⁴⁵, suggests that aptamers can also be used in thisway towards the development of novel DNA-based sensors.

For the applicability of this technology as a practical detectionmethod, the sensitivity of detection must be sufficient. As described,the sensitivity of this system is limited by the affinity of theincorporated aptamer sequence for its target ligand (given that themagnitude of the signal is proportional to the fraction of sensorconstructs bound with ligand). The ATP aptamer described in this Examplepossesses a dissociation constant in the μM range for the adenosineligand. Such a binding affinity would be insufficient for a practicalsensor intended to monitor, for instance, hormone levels in blood (forwhich, sensor-analyte affinities in the low nM to high pM range would berequired). Binding affinities of the nM-pM range, however, are possibleand have been obtained with nucleic acid aptamers; for example, an RNAaptamer selected for binding to the aminoglycoside antibiotictobramycin, possessed a binding constant of 770 pM⁴⁶.

More broadly, the receptor component of such DNA sensors need not initself be composed of DNA or RNA as discussed above. Organic orinorganic hosts, which undergo significant conformational change uponbinding their cognate guest could, in principle, be incorporated inplace of DNA or RNA aptamers into the design of such sensors.

Harnessing the potential of conformational switches in nucleic acids isa relatively new endeavor. It has been used, to date, in the developmentof a mechanical switch⁴⁷, allosteric enzymes⁴⁸ and, now, electronicdevices. The ability to monitor the presence and concentration ofanalytes electrically promises the development of rapid, DNA-based,solid-state detection devices for virtually any compound.

3.1 Example Synopsis

FIGS. 17-23 illustrate an embodiment of the invention for detecting thepresence of the serum protein thrombin. An aptamer that binds thrombinwas selected and incorporated into a coupled ligand DNA sensor that wasbound on a gold chip via detector stem 14′. Ferrocene was covalentlycoupled to the end of reporter stem 16′ to allow for direct electronicdetection of thrombin. Detection of thrombin in the picomolar range wassuccessfully measured, thereby demonstrating that a chip-based sensormay be readily constructed using an aptamer capable of binding thetarget molecule of choice.

3.2 Methods and Materials 3.2.1 Preparation of DNA Constructs andBiochemical Tests

5′-C6-amino-oligonucleotides and other oilgos were purchased from CoreDNA Services Inc. (Calgary, AB) and size-purified using denaturing (50%urea, w/v) polyacrylamide gel electrophoresis (PAGE) before use.Quantification was done on Cary dual beam spectrophotometer using theabsorbance at 260 nm estimated for single stranded DNA.5′-C6-amino-oligonucleotides were used for coupling anthraquinone (AQ)groups in order to determine the pattern of damage induced along thelength of the DNA, for which the procedures were reported in ourprevious publications in detail.⁴⁹ The covalent attachment wasaccomplished by reacting the N-hydroxysuccinimide ester ofanthraquinone-2-carboxylic acid. AQ-oligonucleotides were purified byAgilent HPLC system using an Agilent Zorbax ODS RP-18 5-μm column,eluting with a gradient of 0.1 M triethylammonium acetate (TEAA,pH=7.0)/CH₃CN (20:1, buffer A) and 100% CH₃CN (buffer B) at 1.0 ml/min.To lower the background cleavages of irradiation experiments whichcontained base labile lesions created during chemical synthesis,oligonucleotides to be ³²P-labeled were pretreated with 10% (v/v)piperidine at 90° C. for 30 min, and followed by lyophilization, priorto 5′-labeling with ³²P using standard T4 kinasing protocols and PAGEpurification.

The coupled-ligand DNA constructs shown in FIG. 17 were formed byannealing mixtures of equimolar concentrations (0.125 mM) of constituentstrands in the binding buffer (50 mM Tris, pH 7.4, 50 nM NaCl and 0.002mM EDTA). Samples were heated to 90° C. for 2 min, cooled slowly to roomtemperature, mixed with desired concentrations of MgCl₂, BSA solution(or other non-specific binding proteins) and then kept in ice; in final,ice-cold diluted thrombin was added. Original thrombin (96 mM, kept in−80° C.) was freshly diluted either in storage buffer (50 mM sodiumcitrate, pH 6.2, 0.1% PEG8000 and 50 mM NaCl) or in diluted serumsolution before use. The diluted serum solution was prepared withstandard buffer (50 mM Tris, pH 7.4 and 50 mM NaCl). All solutions wereincubated in a water bath at 37° C. for 5 min before UV irradiation.

Upon the incubation of the DNA construct with the testing protein, thesample was placed under a UVP Black-Ray UVL-56 lamp (365 nm) for 60 minat a distance of 4 cm from the lamp at 4° C. in 50 mM Tris, pH 7.4, 50mM NaCl and 0.02 mM EDTA, in the presence of various thrombinconcentrations. Excitation of the AQ results in one-electron oxidationof the DNA, which generates a base radical cation. Temperature wasmaintained by having the sample tubes placed in a water bath set to thedesired temperature. Following irradiation, the samples wereprecipitated in 70% ethanol solution with 300 mM sodium acetate and thenthe DNA pellets dissolved in 10% piperidine and heated for 30 min at 90°C. as described previously in detail.^(49,50) All samples were thenlyophilized, dissolved in a denatural dye, heated to 100° C., cooled,and loaded on a 12% polyacrylamide sequencing gels. Imaging andquantification of the gels were carried out using a Typhoon 9410Phosphorimager and ImageQuant 5.2 software, by which densitmetry tracesof entire lanes in a gel can be obtained. The density of a G8 peak wastaken as a percentage of total signals within whole lane. The relativesignal of the sensor construct as a function of the thrombinconcentration as plotted in FIG. 19, was quantitated as a ratio of thechange of DNA cleavage (ΔI) at the detector stem guanine triplet(corrected in each case for unirradiated cleavage) in the presence ofthrombin relative to that in the absence of thrombin (I).

3.2.2 DNA-Immobilization and Electrochemical Tests

The synthetic DNA oligonucleotide contains the thrombin aptamer(47-mer), HO—(CH₂)₆—SS—(CH₂)₆—O-5′-TCT CCA GCG TCG AAA GGT TGG TGT GGTTGG TTT AAT CTC GAG CTA AA-3′ (Strand 1), the amino-derivative of theshorter strand (24 mer), H₂N—(CH₂)₆—O-5′TTT AGC TCG AGA CGA CGC TGGAGA-3′ (Strand 2) and its complementary strand, HO—(CH₂)₆—SS—(CH₂)₆-TCTCCA GCG TCG TCT CGA GCT AAA (Strand 3) were purchased from the Core DNAServices Inc. (Calgary, AB). The 5′-thiol modifier was acquired fromGlen Research (Sterling, Va.). The Fc-modification of Strand 2 wascarried out in the same manner as for the AQ-modification, which followsan established protocol.⁵¹

The synthesized oligonucleotides were purified by reverse-phase HPLC ona Gemini 5-μm C18/1110 Å column (Phenomenex, Torrance, Calif.), elutingwith a gradient of 0.1 M triethylammonium acetate (TEAA, pH 7.0)/CH₃CN(20:1, buffer A) and 100% CH₃CN (buffer B) at 1.0 ml/min. In order toreduce the disulfide bonds and generate thiol-terminated single-strandedDNA (HS-ssDNA; Strands 1 and 3), the HPLC purified sample (0.5-2.0 nmol)was treated with 40 μl 10 mM Tris(2-carboxyethyl)phosphine hydrochloride(TCEP, Aldrich-Sigma) in 100 mM Tris buffer at pH 7.5 at roomtemperature for 4 h, followed by desalting with a MicroSpin G-25 Column(Amersham Biosciences, England). The columns were equilibrated withdeoxygenated water prior to use.

For the DNA constructs used to modify gold electrodes (FIG. 5), 5.0 μMof freshly prepared HS-ssDNA (Strand 1 for the sensor or strand 3 forthe control) was hybridized with the 5.5 μM Fc-modified strand (Strand2) in deoxygenated standard buffer (50 mM Tris, pH 7.4 and 50 mM NaCl)at 80° C. for 2 min, followed by slow cooling to room temperature.

Gold substrates (100 nm Au/5 nm Cr/glass) were purchased from EvaporatedMetal Films Inc. (Ithaca, N.Y.). Prior to modification, they werecleaned with freshly prepared piranha solution (3:1 mixture ofconcentrated H₂SO₄ and 30% H₂O₂; WARNING: piranlha solution reactsviolently with organic solvents) at 90° C. for 5 min and then rinsedthoroughly with water. The freshly cleaned gold was modified byspreading a droplet of 10 μl of 5 μM DNA constructs in standard bufferon its surface. The gold chips coated with DNA were stored in a box at100% relative humidity at room temperature, for 12-48 hrs. Aftermodification, they were rinsed with standard buffer incubated in 1 mM6-mercapto-1-hexanol (MCH, Aldrich) in the same buffer for 1 h topassivate the gold surfaces (except where otherwise indicated) andthoroughly rinsed again with the same buffer. Based on biochemicaltests, the electron transfer direction is expected to be from the goldchip to the redox indicator, Fc (ferrocene); Fc must be pre-oxidized toform Fc⁺ in order to accept an electron.

Electrochemical experiments were carried out using a 1-mLthree-electrode cell with a gold chip, modified with the DNA constructs,as the working electrode (with a geometric area of 0.126 cm²). Aplatinum wire counter-electrode and an Ag|AgCl|3 M NaCl referenceelectrode were used. Cyclic voltammetry (CV) and square wave voltammetry(SQW) measurements were performed at ambient temperature (21-23° C.) instandard buffer (50 mM Tris, pH 7.4 and 50 mM NaCl) before and afterincubation in different amount of thrombin (in the same buffer) using aμAutolab II potentiostat/galvanostat (Eco Chemie B. V., Utrecht,Netherlands). The reaction of chips with thrombin was performed in 20 μlthrombin in standard buffer or diluted serum solutions (diluted with 50mM Tris, pH 7.4 and 50 mM NaCl twice or 100 times) at differentconcentrations at 4° C. for 10 min, then washed with standard bufferthree times. Chips were scanned in protein-free buffer from +0.48 V to−0.1 V with precondition at +0.48 V for 5 s.

3.3 Results and Discussion 3.3.1 Deoxyribosensor Design and TestingUsing a Biochemical Protocol

One advantage of a sensor according to this embodiment is the ability tomake use of pre-existing DNA receptors (aptamers) for the binding ofspecified molecular analytes. The DNA aptamer must be functionallycoupled to a double-helical conduction path, such that conformationalchange wrought in the aptamer upon analyte binding results in aconcomitant change in conductivity of the double helical element. As anexample of a sensor according to this embodiment of the invention, achip-based sensor for the plasma protein thrombin was prepared byutilizing an aptamer sequence known to bind thrombin.

In 1992, Toole and co-workers described their isolation of a singlestranded DNA aptamer to the protease thrombin of the blood coagulationcascade and reported binding affinities in the range of 25-200 nM.⁵² Ina subsequent publication,⁵³ Paborsky et al. further demonstrated theaptamer-binding site on thrombin using a solid-phase plate binding assayas well as by chemical modification of the protein. Due to the ease withwhich novel, made-to-order aptamers can be selected from large, randomsequence DNA and RNA libraries, and their generally impressiveselectivity and affinity, they are widely regarded as ideal recognitionelements for biosensor applications.^(18a) In line with this claim,anti-thrombin aptamers have been explored recently as sensing entitiesin conjunction either optical or electrical readouts.⁵⁴⁻⁵⁸ One of thestate-of-the-art designs proposed by Xiao et al. reported nanomolar (nM)detection of thrombin in blood serum; their electronic sensing was basedon what is known to be a large scale, binding-induced, conformationalswitch of the thrombin-binding aptamer.⁵⁶

To construct a sensor capable of detecting the plasma protein, thrombin,the DNA aptamer sequence in vitro selected by Bock et al.⁵² (5′-GGTTGGTGTG GTTGG) was co-opted to construct a series of potential“coupled-ligand” sensors by combining it with double helical “reporter”and “detector” stems used by Sankar et al.⁵⁰ as the conduction path. Thedifferent sensor constructs varied in sequence of the three-way junctionthat serves to join the aptamer to the conduction path are shown in FIG.17. Five different configurations of the junction base-pairs (3WJ-1 to3WJ-5) were examined to select the one showing optimal performance. Thejunctions in these constructs (3WJ-1 to 3WJ-5) incorporate, variously,AA bulges at different locations, in addition to distinct elements ofA-T and G-C base pairs (FIG. 17). In practice, these putativedeoxyribosensors were assembled by complementarity-determinedhybridization of two distinct DNA strands—the first, derivatized at its5′-terminus with anthraquinone (AQ), 3-4 and the second strand,radiolabeled at its 5′-hydroxyl end with a ³²P-phosphoester,incorporating the aptamer sequence.

The prediction in testing these five distinct sensor constructs was thatthe overall design of one or more constructs might encourage thestacking of the “reporter” and “detector” stems concomitant to thebinding of thrombin to the aptamer loop. The design of these differentthree-way junction elements were based, in part, on empirical rulesestablished by Welch et al.⁵⁹ for the construction of regular DNAthree-way junctions (“regular” junctions consist of three fullybase-paired helical stems of DNA, rather than two helical stems and anaptamer loop as shown in FIG. 17). The presence of a two-nucleotidebulge within a “regular” junction typically leads to preferentialstacking of two of the three component helices. However, the stackingrules strictly hold only where three regular helices (along with theunpaired two-nucleotide bulge) constitute the three-way junction. Weenvisioned that replacement of one of the three helical arms by a veryshort stem of two base-pairs terminating in an aptamer loop might resultin the disruption of stacking between the two stems, which stackingmight be restored upon tightening of the aptamer loop and adjoining 2-bpstem by the binding of thrombin to the aptamer.

The above designs were first evaluated biochemically by studying theeletrophoretic patterns and intensities of charge-flow generated guanineoxidative damage (determined by piperidine-catalyzed DNA cleavage)within them, notably at a specific “trapping” sequence³³ consisting ofthree consecutive guanines (marked as “D” in FIG. 17) within the“detector” helix. The constructs from 3WJ-1 to 3WJ-5 were eachirradiated at 366 nm to set up charge flow within them. Experiments werecarried out in a test buffer (50 mM Tris, pH 7.4, 5 mM KCl, 50 mM NaCland 0.02 mM EDTA), with or without added thrombin. Irradiated sensorsamples (as well as unirradiated, i.e., “dark” controls) were thentreated with 10% (v/v) piperidine at 90° C., lyophilized to dryness, andtheir fragmentation patterns analyzed by denaturing PAGE. FIG. 18 showsthe data for each construct, the “d” lane shows a “dark” control (noirradiation), where the deoxyribosensor was incubated in the dark in thepresence of 160 nM thrombin; the “w” lane shows irradiated samples inthe absence of protein, and the “Th” lanes show duplicate experiments onirradiated sensors in the presence of 160 nM thrombin. Reference Gladders are shown to the left for each of the constructs. It can be seenthat the different sensor constructs showed distinct oxidative cleavagepatterns, especially at the detector guanine triplet (marked as “D” inFIG. 18). It can be seen that with both the constructs 3WJ-3 and 3WJ-4,notably higher band intensities were seen in the “Th” (UV irradiationplus added thrombin) lanes, relative to the “d” (no irradiation butadded thrombin) and “w” (irradiation but no added thrombin) controls. Ofthe two constructs, 3WJ-3 provided the highest signal at the “D”sequence in response to the presence of thrombin, and this sensorconstruct was carried forward for further studies.

In addition, buffer solutions and temperature were systematically variedfor the 3WJ-3 sensor, to determine the optimal conditions for itsdetection of thrombin. We found a superior response when sensorirradiation was carried out at 4° C. rather than at ambient (25° C.) orphysiological (37° C.) temperatures. At 10 mM Mg²⁺, the addition ofmonocations (Na⁺ and Li⁺) ranging from 50 to 150 mM had no effect, whilein comparison without salt, low concentration of salt was necessary formaintaining the sensor construct. Different Mg²⁺ concentrations (0, 5,10, 15 20 mM) were performed; increasing the Mg²⁺ concentration did nothelp to increase the sensor response. The presence of bovine serumalbumin (BSA) at 0.01% (w/w) in the buffer, nevertheless, improved thesensor performance by significantly reducing background DNA cleavage,presumably by discouraging the non-specific binding of thrombin to thesensor.

The inventors carried out the study of relative strand cleavage at the“D” sequence as a function of the concentration of added thrombin (underthe optimized solution and temperature conditions, see Section 2.3Methods and Materials above). FIG. 19 shows that the sensor signal (theincrease in intensity seen in the “D” sequence, as determined byphosphorimagery, normalized to the background intensity, ΔI/I) showed amonotonic increase initially and saturated when the thrombinconcentration rose to ˜160 nM. The dynamic range of the detection andthe sensitivity demonstrated in FIG. 19 are comparable with thosepreviously demonstrated with other optical protocols.^(58,58) Moreimportantly, control experiments with other proteins such as avidin,IgA, and IgG, all at fairly high concentrations (160 nM), showedinsignificant sensor signals, which confirmed that the specificity ofthe designed 3WJ-3 deoxyribosensor was indeed for thrombin.

3.3.2 Chip-Based Sensor Design and an Electrochemical Thrombin Assay

Following the biochemical demonstration of thrombin detection using a“coupled-ligand” sensor, 3WJ-3, the inventors constructed a version ofthe sensor that might be appropriate for chip-based thrombin detection,via a direct electrochemical readout. FIG. 20( a) shows such a sensor,whose sequence was adapted from 3WJ-3 (notably, the reporter/AQ stem wasshortened and the guanine triplet in the detector stem eliminated tofacilitate electron-transfer from electrode to the redox center).Instead of an electron acceptor, such as the anthraquinone (AQ) used inthe biochemical experiments, ferrocene (Fc) was covalently tethered tothe terminus of the reporter stem as a redox indicator. Furthermore, thedistal end of the construct was modified with a thiol group, whichenables the formation of robust DNA monolayers on a gold electrode via asulfur-gold linkage.^(60,61) Upon the binding of thrombin to the aptamerportion of the sensor, the induced switch in the conduction pathway maybe measured directly with conventional electrochemical measurements.

Before testing the performance of the thrombin chip-bound sensor itself,the electrochemical behavior of a control Fc-terminated DNA double-helix(duplex) immobilized on a gold chip (FIG. 20( b)) was investigated. Forthe purpose of comparison, the sequence in this double helix wasidentical to the conduction path within the deoxyribosensor (FIG. 20(a)), but lacking the aptamer loop and stem. Cyclic voltammetry (CV) andsquare wave voltammetry (SQW) were both carried out as parts of theinitial investigation. Both experiments showed that the reductioncurrent was more pronounced than the oxidation current (see supportinginformation). For CV, the reduction current at first scan was largerthan that at later scans; furthermore, the redox reaction was morepronounced at lower scan rate (0.1 V/s) than at a higher scan rate (5V/s). These results imply that the electron-transfer in the immobilizedduplex may prefer one direction (from the gold chip to the ferriciniumion (Fc⁺) at oxidation state over the other (from the Fc, i.e., reducedstate, to the gold chip). It has been shown experimentally that SQW ismore sensitive than CV, therefore, SQW along a reduction sweep wasapplied to further characterize the DNA constructs.

FIG. 21 shows the SQW data measured with gold electrodes modified withthe sensor constructs shown in FIG. 20( a). The sensor signal (nowmeasured directly as an electrical current) increased significantly uponincubation of the sensor chip with thrombin at picomolar (pM)concentrations in the standard buffer solution (50 mM Tris, pH 7.4 and50 mM NaCl). FIG. 22 shows the dependence of the sensor signal onthrombin concentrations. The sensor signal was distinct at aconcentration as low as 5 pM and increased with increasing thrombinconcentrations (up to 200 pM). FIG. 22( b) shows the linear range ofresponse (between 5 and 100 pM) and also includes, for comparison, theresponses measured in the presence of other proteins such as BSA (67KDa, pI=4.8), Avidin (68 KDa, pI=10.5), IgA (350 KDa, pI=3.5-5.5), andIgG (150 KDa, pI=5.5-9). Three of the above proteins are abundant in theserum. Even with concentrations as high as 80 pM, no evidence forbinding of these non-cognate proteins to the sensors were detected,relative to the clear signal seen in the presence of thrombin.

The results shown in FIG. 22 not only demonstrate a successfultranslation of the “coupled-ligand” sensor to a chip-based device withdirect electronic readout, but also show an unprecedented improvement insensitivity. Moving from the solution-based biochemical tests tochip-based electrochemical detection, sensitivity was enhanced from thenM range (which is at the same level as reported by others⁵⁵⁻⁵⁸) to thepM range. This may be partially due to a reduction in the backgroundsignal, which is always present at a fairly high level in the DNAfragmentation and gel electrophoresis experiments. The major factor inthe sensitivity improvement, however, may be the use of a limited numberbut readily accessible sensor constructs on the electrode, given thatthe surface density of the DNA constructs on the gold chip is only 2.6pmol/cm² (determined by CV measurements in the presence of 5.0 M[Ru(NH₃)₆]³⁺ redox cations^(61,62)). The electrode surface area issmall, at 0.126 cm², and it is clear that even pM concentrations ofthrombin in the solution are sufficient to activate the inunobilizedsensor population. The dissociation constant, K_(d), for thethrombin-aptamer interaction has been shown to be as low as 2.7nM,^(52,57) which is consistent with the data presented herein.

In retrospect, reports of electrochemical probing of conductivitychanges in DNA constructs are limited;^(44,51,63) Kelley et al. testedfor lateral charge propagation through double-helical DNA films, andfound that the electrochemical signals decreased linearly withincreasing percentages of mutated duplexes (i.e., containing C-Amismatches).⁴⁴ Wong and Gooding have recently shown that a long-rangecharge transfer approach can be used to probe cisplatin-induced DNAperturbations,⁶³ where differences in the charge flow could beattributed to changes in conformation of the DNA duplexes. The inventorshave provided evidence that structural changes to a DNA aptamer elementlocated adjacent to a “disrupted” conduction path (two poorly stackingDNA double helices) helps to re-establish the stacking and hence“repair” the conduction path, a phenomenon that had so far been inferredonly indirectly, from biochemical experiments.^(49,50) Interestingly,the structural changes induced in this particular aptamer upon thrombinbinding has also been used in the methodology of Xiao et al.,⁵⁶ whichdepends on altered distances between the redox center and the electrodesurface upon ligand binding. However, the sensitivity observed in thatreport was 6.4 nM, and the dynamic range was from 6.4 to 768 nM.Differences in design between the sensors of the present invention andthe sensor construct of Xiao et al.⁵⁶ may be responsible for theobserved differences in sensitivity and dynamic range of detection.

While the experiments reported above were carried out in theclose-to-physiological standard buffer, the inventors also examined theperformance of the thrombin sensor in the presence of blood serum. Giventhe very high sensitivity of this sensor (detecting in the pM range ofanalyte), practical application of such electronic sensors wouldtypically be made only where blood serum (or other body fluids) havebeen diluted by thousands of times. In fact, the physiologicalconcentrations of thrombin in resting and activated blood range from lownM to low μM.⁶⁴ When incubations and current measurements were made withpM concentrations of thrombin in diluted serum (dilutions made with thestandard buffer), no significant influence of serum on the sensor signalwere found at serum dilutions of 100-fold (shown as hollow circles inFIG. 23) and higher. The slightly higher current measurements made inthis case may even reflect levels of endogenous thrombin originally inthe serum.⁵⁶ In one experiment, pM concentrations of thrombin were firstincubated with the immobilized deoxyribosensor in the standard buffer,following which the buffer was removed and replaced with undilutedserum, prior to the measurement of current. This set of data, shown astriangles in FIG. 23, indicates that it is still possible to getquantitative response from the sample, although the undiluted serum doesinfluence the sensor signal to some extent, probably owing to the veryhigh overall concentration of proteins in undiluted serum. However, thisresult clearly demonstrates that the effect of non-specific proteinsand/or electrolytes in the original body fluid on this new sensorapplication may actually be very small.

This example demonstrates the direct electrical detection of abiomedical analyte, thrombin, using a unique design of a DNA switch,namely a “deoxyribosensor”. The inventors have designed a number of suchsensors to demonstrate that charge transfer through a double-helical DNAconduction path can be modulated by conformational changes within it,resulting from the adaptive binding of a specified ligand/analyte to aDNA aptamer functionally appended to the conduction path. Such ameasurable change in charge transfer concomitant to ligand bindingresults from both large and relatively subtle conformational changes inthe aptamer upon ligand binding.^(49,50) The change in charge transferthrough the sensor can be measured biochemically in solution, andelectrochemically on a chip. Specifically, a sensor originally designedfor a solution-based sensing of a plasma protein, thrombin, can be“translated” to a chip-based electronic device, which detects andquantitates as low as picomolar concentrations of thrombin in both adefined buffer solution as well as in diluted blood serum. Owing to thegeneric and modular nature of the design of the sensors (coupling a DNAconduction path and an aptamer loop) and the established surfacechemistry used to construct the biochip that utilizes the sensor, thisembodiment of the invention is widely applicable to the development ofrapid, DNA-based electronic sensors for any number of small andmacromolecular analytes of biomedical and/or environmental importance.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the scope thereof.Accordingly, the scope of the invention is to be construed in accordancewith the substance defined by the following claims.

REFERENCES

-   1. Giese, B. Acc. Chem. Res. 2000, 33, 631-636.-   1a. Famulok, M. Nat. Biotechnol. 2002, 20, 448-449.-   1b. Campàs, M.; Katakis, I. Trends Anal. Chem. 2004, 23, 49-62.-   2. Schuster, G. B. Acc. Chem. Res. 2000, 33, 253-260.-   3. Kelly, S. O.; Holmlin, R. E.; Stemp, E. D. A.; Barton, J. K. J.    Am. Chem. Soc. 1997, 119, 9861-9870.-   4. Giese, B.; Wessely, S. Angew. Chem. Int. Ed. 2000, 39, 3490-3491.-   5. Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.;    Barton, J. K. Nature Biotech. 2000, 18, 1096-1100.-   6. Hall, D. B.; Barton, J. K. J. Am. Chem. Soc. 1997, 119,    5045-5046.-   7. Rajski, S. R.; Kumar, S.; Roberts, R. J; Barton, J. K. J. Am.    Chem. Soc. 1999, 121, 5615-5616.-   8. Rajski, S. R.; Barton, J. K. Biochemistry 2001, 40, 5556-5564.-   9. Gasper, S. M.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119,    12762-12771.-   10. Kan, Y.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121,    10857-10864.-   11. Porath, D., Bezryadin, A. & de Vries, S. (2000) Nature 403,    635-638.-   12.a Fink, H.-W. & Schonenberger, C. (1999) Nature 398, 407-   12.b Okahata, Y., Kobayashi, T., Tanaka, K. &    Shimomura, M. (1998) J. Am. Chem. Soc. 120, 6165-6166.-   13. Gasper. S. M. & Schuster, G. B. (1997) J. Am. Chem. Soc. 119,    12762-12771.-   14. Hall, D. B., Holmlin, R. E., & Barton (1996) Nature. 382,    731-735.-   15.a Nunez, M. E., Hall, D. B., & Barton (1999) Chem. Biol. 6,    85-97.-   15.b Bixon, M. Giese, B., Wessely, S., Langenbacher, T.,    Michel-Beyerle, M. E. & Jortner, J. (1999) Proc. Natl. Acad. Sci.    USA 96, 11713-11716.-   16. Saito, I., Takayama, M., Sugiyama, H. Nakatani, K., Tsuchida, A.    & Yamamoto., M. (1995) J. Am. Chem. Soc. 117, 6406-6407.-   17. Gold. L.; Polisky, B.; Uhlenbeck, O.; Yarus, M. Annu. Rev.    Biochem. 1995, 64, 763-797.-   18. Herman, T.; Patel, D. J. Science 2000, 287, 820-825.-   18a. Tan, W. H.; Wang, K. M.; Drake, T. J. Curr. Opin. Chem. Biol.    2004, 8, 547-553.-   19. Rajski, S. R. et al. (1999) J. Am. Chem. Soc. 121, 5615-5616.-   20. Aich, P., Labiuk, S. L., Tari, L. W., Delbaere, L. J.,    Roesler, W. J., Falk, K. J., Steer, R. P., Lee, J. S. (1999) J. Mol.    Biol. 294, 477-85.-   21. Boon, E. M., Ceres, D. M., Drummond, T. G., Hill, M. G. &    Barton, J. K. National Biotechnol. 18, 1096-1100, 2000.-   22. Peattie, D. A. (1979) Proc. Natl. Acad. Sci. USA 76, 1760-1764.-   23. Wang, W. K., et al. (2000) J. Microbiol. Immunol. Infect. 33,    131-40.-   24. Puglisi, J. D., Tan, R., Calnan, B. J., Frankel, A. D., &    Williamson, J. R. (1992) Science 257, 76-80.-   25. Battiste J. L., Tan, R., Frankel, A. D.,    Williamson, J. R. (1994) Biochemistry 33, 2741-7-   26. Odom, D. T. & Barton, J. K. (2001) Biochemistry 40, 8727-8737.-   27. Saito, I. & Kino, K. (1998) J. Am. Chem. Soc. 120, 7373-7374.-   28. Huizenga, D. E.; Szostak, J. W. Biochemistry 1995, 34, 656-665.-   29. Lin, C. H.; Patel, D. J. Chem. Biol. 1997, 4, 817-832.-   30. Odom, D. T.; Dill, E. A.; Barton, J. K. Chem. Biol. 2000, 7,    475-481.-   31. Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L.;    Chan, K. J. Am. Chem. Soc. 1989, 111, 7226-7232.-   32. http://www.probes.com/media/pis/mp00143.pdf-   33. Hall, D. B.; Holmlin, R. E.; Barton, J. K. Nature 1996, 382,    731-735.-   34. Nunez, M. E.; Hall, D. B.; Barton, J. K. Chem. Biol. 1999, 6,    85-97.-   35a. Henderson, P. T.; Jones, D.; Hampikian, G.; Kan, Y.;    Schuster, G. B. Proc. Natl. Acad. Sci. USA 1999, 96, 8353-8358.-   35b. Grinstaff, M. W. Agnew. Chem. Int. Ed. 1999, 38, 3629-3635.-   36. Saito, I.; Nakamura, T.; Nakatani, K.; Yoshioka, Y.; Yamaguchi,    K.; Sugiyama, H. J. Am. Chem. Soc. 1998, 120, 12686-12687.-   37. Sanii, L.; Schuster, G. B. J. Am. Chem. Soc. 2000, 122,    11545-11546.-   38. Wellinger, R.; Sen, D. Eur. J. Cancer 1997, 33, 735-749.-   39 Simonsson, T. Biol. Chem. 2001, 382, 621-628.-   40. Giese, B.; Amaudrut, J.; Kohler, A. K.; Spormann, M.; Wessely,    S, Nature 2001, 412, 318-320.-   41. Nakatani, K.; Dohno, C.; Saito, I. J. Am. Chem. Soc. 2000, 122,    5893-5894.-   42. Saito, I.; Takayama, M.; Sugiyama, H.; Nakatani, K.; Tsuchida,    A.; Yamamoto, M. J. Am. Chem. Soc. 1995, 117, 6406-6407.-   43. Szalai, V.; Thorp, H. H. J. Am. Chem. Soc. 2000, 122, 4524-4525.-   44. Kelly, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Angew.    Chem. Int. Ed. Eng 1.1999, 38, 941-945.-   45. Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.;    Barton, J. K. Nature Biotech. 2000, 18, 1096-1100.-   46. Wang, Y.; Killian, J.; Hamasaki, K.; Rando, R. R. Biochemistry    1996, 35, 12338-12346.-   47. Mao, C.; Sun, W.; Shen, Z.; Seeman, N. C. Nature 1999, 397,    144-146.-   48. Soukup, G. A.; Breaker, R. R. Curr. Opin. Struct. Biol. 2000,    10, 318-325.-   49. Fahlman, R. P.; Sen, D. J. Am. Chem. Soc. 2002, 124, 4610-4616.-   50. Sankar, C. G.; Sen, D. J. Mol. Biol. 2004, 340, 459-467.-   51. Ihara, T.; Maruo, Y.; Takenaka, S.; Takagi, M. Nucleic Acids    Res. 1996, 24, 4273-4280.-   52. Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.;    Toole, J. J. Nature 1992, 355, 564-566.-   53. Paborsky, L. R.; McCurdy, S, N.; Griffin, L. C.; Toole, J. J.;    Leung, L. L. K. J. Biol. Chem. 1993, 268, 20808-20811.-   54. Ho, H.-A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384-1387.-   55. Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem.    Soc. 2004, 126, 11768-11769.-   56. Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew.    Chem. Int. Ed. 2005, 44, 5456-5459.-   57. Zhu, H.; Suter, J.; White, I. M.; Fan, X. Sensors 2006, 6,    785-795.-   58. Bini, A.; Minunni, M.; Tombelli, S.; Centi, S.; Mascini, M.    Anal. Chem. 2007, 79, 3016-3019.-   59. Welch, J. B.; Walter, F.; Lilley, D. M. J. J. Mol. Biol. 1995,    251, 507-519.-   60. Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am.    Chem. Soc. 1998, 120, 9787-9792.-   61. Yu, H. Z.; Luo, C. Y.; Sankar, C. G.; Sen, D. Anal. Chem. 2003,    75, 3902-3907.-   62. Ge, B.; Huang, Y.-C.; Sen, D.; Yu, H. Z. J. Electroanal. Chem.    2007, 602, 156-162.-   63. Wong, E. L. S.; Gooding, J. J. J. Am. Chem. Soc. 2007, 129,    8950-8951.-   64. Lee, M.; Walt, D. Anal. Biochem. 2000, 282, 142-146.

1. An analyte sensor comprising: (a) a first oligonucleotide stem; (b) asecond oligonucleotide stem; and (c) a receptor site capable of bindingsaid analyte, wherein said receptor site is operatively connected tosaid first oligonucleotide stem and said second oligonucleotide stem,wherein said sensor is alterable between a first conformational statesubstantially impeding charge transfer between said first and secondstems and a second conformational state permitting charge transferbetween said first and second stems, wherein said sensor switchesbetween said first conformational state and said second conformationalstate when said analyte binds to said receptor site.
 2. The sensor asdefined in claim 1, wherein said charge is transferred between saidfirst and second stems through said receptor site in said secondconformational state.
 3. The sensor as defined in claim 1, wherein saidreceptor site is removed from a conduction path between said first andsecond stems in said second conformational state.
 4. The sensor asdefined in claim 1, wherein said sensor switches from said firstconformational state to said second conformational state when saidanalyte binds to said receptor site.
 5. The sensor as defined in claim1, wherein said sensor switches from said second conformational state tosaid first conformational state when said analyte binds to said receptorsite.
 6. The sensor as defined in claim 1, wherein said receptor site isselected from the group consisting of nucleic acids and proteins.
 7. Thesensor as defined in claim 6, wherein said receptor site comprises anucleic acid aptamer selected for binding affinity to a target analyte.8. The sensor as defined in claim 1, wherein said receptor site iscapable of binding to analytes which do not ordinarily bind to DNA. 9.The sensor as defined in claim 1, wherein said first and second stemseach comprise an ordered sequence of nucleotide base pairs, and whereinsaid sensor comprises a switch region at the junction between said firstand second stems, wherein spacial stacking of said first and secondstems is said switch region substantially impedes charge transferbetween said first and second stems in said first conformational state.10. The sensor as defined in claim 9, wherein said switch regioncomprises unpaired nucleotides in said first conformational state. 11.The sensor as defined in claim 10, wherein said unpaired nucleotides arenon-Watson-Crick nucleotides.
 12. The sensor as defined in claim 10,wherein the spacial stacking of said first and second stems within saidswitch region is altered when said sensor switches between said firstand second conformational states.
 13. The sensor as defined in claim 12,wherein said switch region is located proximate to said receptor site.14. The sensor as defined in claim 12, wherein switch region comprisessaid receptor site.
 15. The sensor as defined in claim 9, wherein saidfirst and second stems each comprise a multi-stranded DNA helix.
 16. Thesensor as defined in claim 15, wherein said helix is disrupted in saidswitch domain in the vicinity of said receptor site in said firstconformational state.
 17. The sensor as defined in claim 1, furthercomprising a third oligonucleotide stem comprising said receptor site.18. The sensor as defined in claim 17, wherein said first, second andthird stems are connected together at a three-way junction.
 19. Thesensor as defined in claim 18, wherein at least one of said first,second and third stems comprises a non-Watson-Crick base pairing in thevicinity of said three-way junction.
 20. The sensor as defined in claim17, further comprising a fourth oligonucleotide stem, wherein saidfirst, second, third and fourth stems are connected together at afour-way junction.
 21. The sensor as defined in claim 1, furthercomprising a charge flow inducer coupled to one of said first and secondstems for triggering charge flow in at least one of said first andsecond stems.
 22. The sensor as defined in claim 21, wherein said chargeflow inducer comprises an excitable moiety alterable between anunexcited and an excited state.
 23. The sensor as defined in claim 22,wherein said moiety is an oxidizing agent in said excited state.
 24. Thesensor as defined in claim 22, wherein said moiety is a reducing agentin said excited state.
 25. The sensor as defined in claim 22, whereinsaid moiety is selected from the group consisting of anthraquinone andrhodium (III).
 26. The sensor as defined in claim 1, further comprisinga detector electrically coupled to said first stem for directlymeasuring said charge transfer.
 27. The sensor as defined in claim 26,wherein said detector comprises a conductor.
 28. The sensor as definedin claim 26, wherein said detector comprises a semi-conductor chip. 29.The sensor as defined in claim 1, wherein said receptor site bindsadenosine analyte.
 30. A nanoelectronic chip comprising a plurality ofsensors as defined in claim
 1. 31. A sensor for detecting first andsecond analytes comprising: (a) a first oligonucleotide stem; (b) asecond oligonucleotide stem; (c) a first receptor site capable ofbinding said first analyte; and (c) a second receptor site capable ofbinding said second analyte, wherein said first and second receptorsites are operatively connected to said first and second oligonucleotidestems, wherein said sensor is alterable between a first conformationalstate substantially impeding charge transfer between said first andsecond stems and a second conformational state permitting chargetransfer between said first and second stems, wherein said sensorswitches between said first conformational state and said secondconformational state when said first analyte binds to said firstreceptor site and said second analyte concurrently binds to said secondreceptor site.
 32. A method for detecting the presence of an analytecomprising: (a) providing a sensor as defined in claim 1 having areceptor capable of binding to said analyte; (b) inducing a charge flowin one of said first and second stems of said sensor; and (b) detectingany change in charge transfer between said first and second stems uponbinding of said analyte to said receptor.
 33. The method as defined inclaim 32, wherein the step of detecting any change in charge transfercomprises electrically coupling a detector to the other of said firstand second stems of said sensor.
 34. The method as defined in claim 32,wherein the step of inducing a net charge comprises: (a) coupling amoiety to said second stem alterable between an unexcited and an excitedstate; and (b) exciting said moiety to form an oxidizing agent.
 35. Themethod as defined in claim 34, wherein the step of detecting any changein charge transfer comprises testing for the formation of oxidationproducts of said sensor.
 36. The method as defined in claim 35, whereinsaid testing comprises: (a) heating said sensor in the presence ofpiperidine; and (b) separating any reaction products of step (a) by gelelectrophoresis.
 37. A chip-bound analyte sensor comprising: (a) a firstoligonucleotide stem; (b) a second oligonucleotide stem coupled to saidchip at a first end of said second oligonucleotide stem; and (c) areceptor site capable of binding said analyte, wherein said receptorsite is operatively connected to a first end of said firstoligonucleotide stem and to a second end of said second oligonucleotidestem, wherein said sensor is alterable between a first conformationalstate substantially impeding charge transfer between said first andsecond stems and a second conformational state permitting chargetransfer between said first and second stems, wherein said sensorswitches between said first conformational state and said secondconformational state when said analyte binds to said receptor site. 38.The chip-bound sensor as defined in claim 37 further comprising a redoxindicator coupled to a second end of the first oligonucleotide stem. 39.The chip-bound sensor as defined in claim 37 comprising a plurality ofanalyte sensors coupled to said chip.
 40. The chip-bound sensor asdefined in claim 39 wherein an electrical current is produced when saidanalyte binds to said receptor site.
 41. The chip-bound sensor asdefined in claim 39 wherein said receptor site comprises a DNA aptameradapted to bind thrombin.
 42. A method of detecting an analyte using achip-bound sensor, the method comprising: (a) coupling a plurality ofsensors as defined in claim 1 having a receptor capable of binding tosaid analyte to a chip by covalently attaching said secondoligonucleotide stem to the chip; (b) coupling a redox indicator to afree end of said first oligonucleotide stem; (c) applying a solution tobe tested to the chip; and (d) directly measuring an electrical currentproduced by the binding of the analyte to the receptor site.
 43. Amethod according to claim 42 comprising providing a plurality of sensorsas defined in claim 1 having a receptor capable of binding to thrombinand measuring the electrical current produced by the binding of thethrombin to said receptor site.